Klebsiella pneumoniae, which was first reported in 1882, is an important opportunistic pathogen causing various infectious diseases, including pneumonia, urinary tract infections, bacteremia, and liver abscesses (Hu et al., 2020; Xu et al., 2021). A key characteristic of K. pneumoniae is its ability to acquire new genetic materials (Namikawa et al., 2023). Based on virulence gene carriage, two distinct pathotypes - classical K. pneumoniae and hypervirulent K. pneumoniae (hvKP), are currently spreading worldwide, with each type posing a unique challenge to clinicians (Martin, 2018). Although hvKP has attracted considerable interest in the clinical field, its diagnosis relies on sequencing technology or polymerase chain reaction (PCR), which are seldom available in conventional laboratories. In contrast, a string test, in which an inoculation loop is used to generate sticky strings from a K. pneumoniae colony, is a simple procedure that can be easily performed in conventional laboratories, and it can be utilized to classify K. pneumoniae strains into hypermucoviscous K. pneumoniae (hmKP, positive result in string test) and non-hypermucoviscous K. pneumoniae (non-hmKP, negative result in string test) (Jiang et al., 2022; Wang et al., 2022). Although studies have suggested that hvKP strains often exhibit a hypermucoviscous phenotype (i.e., hmKP) (Martin, 2018; Zhang S, Zhang et al., 2019), the two are not entirely synonymous. The diagnosis of hvKP is based on the presence of virulence genes such as rmpA, iuc, or peg-344, while hmKP is defined phenotypically by the string test.

The present study aimed to systematically investigate the overall clinical and molecular characteristics of hmKP infection in Yantai, China, from the following perspectives: (1) easy implementation of the diagnostic test for hmKP in conventional laboratories, (2) rapid and more efficient detection of hmKP infection, and (3) improvement of patient prognosis through early diagnosis and clinical treatment.

In recent years, the incidence of K. pneumoniae infection has drastically increased (Zhang S, Zhang et al., 2019; Walker, 2020; Zhang et al., 2020; Nakamura-Silva et al., 2022; Le et al., 2023). In many global regions, K. pneumoniae has become the prevalent pathogen with the highest isolation rate (Zhang S, Zhang et al., 2019; Gan et al., 2020; Spadar et al., 2021). Because of its high isolation rate and strong pathogenicity, K. pneumoniae, particularly hvKP strains, has increasingly attracted the attention of researchers worldwide (Marr, 2019; de Oliveira Franco et al., 2024). The hvKP strain is highly pathogenic, with a high mortality rate and a high risk of dispersal. However, the diagnosis of hvKP strains relies on sequencing or PCR technology, which is expensive, time-consuming, and difficult to implement in conventional laboratories (Russo et al., 2018). In contrast, the diagnostic procedure for hmKP and non-hmKP strains is rapid, simple, and economical (Dentice Maidana et al., 2023; Jin et al., 2023; Campanero-Rhodes et al., 2024). Moreover, it could aid clinical practice and therefore has a high practical value.

Traditionally, hmKP infection has been predominantly associated with liver abscesses (Choby et al., 2020; Valente-Acosta et al., 2022). In the present study, however, we found hmKP to be highly prevalent, accounting for 18.1% of all K. pneumoniae infections, with liver abscess cases constituting merely 25% of hmKP-infected patients. We detected hmKP strains in almost all specimen types and in patients receiving treatment in different departments, with a wide age range. A high proportion of ICU patients with hmKP infection and high levels of inflammatory indicators (WBC count and CRP and PCT levels) indicated the high pathogenicity of the isolated hmKP strains. Similar to hvKP, hmKP infection is also common in diabetes patients. In the present study, diabetes patients accounted for 39.8% of the patients with hmKP infection.

The phylogenetic tree showed that K1 and K64, K64 and K22, and K23 and K30 were from the same branch, indicating that these serotypes evolved from the same clone. Similarly, different sequence types on the same branch showed phylogenetic relationships, such as ST23 and ST5214, ST65 and ST375, and ST11 and ST34; moreover, some phylogenetic relationships were reported in the literature (Sanikhani et al., 2021; Michael et al., 2023; Jiang et al., 2024). Among the isolated hmKP strains, the capsular serotypes K1/K2 and sequence types ST23/ST65 were predominant, and their distribution was similar to those reported for hvKP, which indirectly indicated that a major proportion of the isolated hmKP strains was hvKP. Previous studies (Jin et al., 2021; Wang et al., 2023; García García B, Fernández-Manteca et al., 2024) have established correlations between specific capsule serotypes and sequence types, notably K1 with ST23/ST700 and K2 with ST65/ST25. Our findings not only confirm these associations but also reveal novel, strong pairings. The most striking is the K54/ST29 association (OR = 865.00), suggesting a previously under-characterized clone of potential importance. Other notable novel associations include K57/ST412 and K20/ST893. Although statistically significant (p < 0.05), the associations for K1/ST700 and K2/ST380 showed very wide confidence intervals (extending near 1), indicating low precision due to limited sample sizes. Therefore, these specific findings require validation in larger cohorts.

In July 2024, the World Health Organization issued a precautionary report stating the global spread and risk of hvKP infection. The hvKP strain, particularly ST23 type, can cause fatal infections even in immunocompetent populations due to its drug resistance property. In the present study, 76 hvKP strains were detected among the 88 hmKP isolates, accounting for 86.4% of the total isolates. Among these 76 hvKP strains, 28 strains were ST23 type hvKP, indicating that the ST23 type hvKP strain is highly prevalent in hmKP isolates. Moreover, 25% (n = 7) ST23 type hvKP strains were resistant to third-generation cephalosporins. In particular, a ST23 type HCKP strain was detected. Another ST23 type hvKP strain was sensitive to carbapenems but carried the bla_KPC-2 resistance gene. These findings presented new challenges to combat this harmful pathogen. Because of the simultaneous occurrence of high virulence and antibiotic resistance, we expect that the risk of transmission of such strains will remarkably increase. The transmission or outbreak of such pathogenic strains in hospitals could lead to a disastrous outcome; hence, it is very critical to take necessary steps to prevent the spread of these highly pathogenic strains.

An important characteristic of K. pneumoniae is its ability to carry multiple virulence factors, including capsule, lipopolysaccharides, pili, siderophores, allantoin utilization, outer membrane proteins, and type 6 secretory system (Ombada et al., 2023; Park, 2023; Ragheb, 2024; Wang et al., 2024). Among these, capsule and siderophores are the primary virulence factors (Wang et al., 2020; Cardoso Almeida et al., 2024). The virulence genes rmpA and rmpA2 regulate the synthesis of the extracellular polysaccharide capsule and are responsible for the hypermucoviscous phenotype (Kim et al., 2022; Yomogida D, Kuwano et al., 2024), as evidenced by their high prevalence in hmKP strains. K. pneumoniae expresses four types of siderophores: aerobactin, enterobactin, salmochelin, and yersiniabactin, with aerobactin being the most predominant one (Marr, 2019; Le et al., 2022). The relevant virulence determinants mainly include the iuc, ent, iro, and ybt genes encoding aerobactin, enterobactin, salmochelin, and yersiniabactin, respectively (Martin, 2018; Yang Y, Yang et al., 2021; Tanimoto et al., 2022). As shown previously, aerobactin is often concomitant with hypermucoviscosity (Choby et al., 2020), which was also validated in the present study. In this study, the isolated hmKP strains commonly carried the virulence determinants such as rmpA, rmpA2, iuc, iro, peg-344, and clb, indicating the strong virulence of these isolates. mrk is involved in the formation of pili and plays an important role in biofilm formation (Tada et al., 2023); strains carrying mrk can easily cause infections of indwelling catheters and drainage tubes (Russo et al., 2018). In the present study, mrk was detected in almost all K. pneumoniae strains, indicating that patients with indwelling catheters and drainage tubes should be cautious about K. pneumoniae infection development.

In the present study, although several hmKP strains were susceptible to the tested antibiotics, we should remain cautious about the emergence of multidrug-resistant (MDR) hmKP strains. Here, we detected 8 MDR hmKP strains, implying that these strains were resistant to three or more types of antibiotics. Especially the two cases of HCKP in this study, which pose a huge challenge for clinical diagnosis and treatment. Therefore, early identification, treatment, and prevention of the spread of such pathogens are highly critical steps to prevent future epidemics associated with these strains.

We also found that the detected resistance genes and antibiotic resistance were not completely consistent. The detection of resistance genes does not necessarily imply resistance to the specific antibiotic. Similarly, antibiotic resistance does not indicate the presence of resistance genes; this phenomenon is well explained in the results for quinolone and β-lactam resistance genes and antibiotic susceptibility. The oqxA and oqxB genes encoding quinolone resistance were present in almost all strains; however, the resistance rates to quinolone antibiotics (ciprofloxacin and levofloxacin) were relatively low and did not exceed 30%.

Although this study presents the epidemiological characteristics of hmKP infection, it has some limitations. First, this was a single-center study; therefore, the results may not be fully representative of the global scenario. Second, the sample size was relatively small, which may have resulted in biased findings. Nevertheless, the results of the present study contribute to the effective identification of hmKP infection and provide insights into the molecular characteristics of hmKP strains; these findings could aid clinical practice and facilitate the rapid and effective diagnosis of patients with hmKP infection.

HmKP infection is prevalent in Yantai, China, with dominant K1/ST23 and K2/ST65 clones carrying prevalent virulence genes. Notably, HCKP strains were detected locally. Enhanced vigilance against such high-risk strains is urgently needed to guide local clinical response and infection control.