As the principal cause of hospital-acquired pneumoniae leading to multiple organ failure and even death, Klebsiella pneumoniae (Kp) has become an increasingly common and highly concerned pathogen in recent years (Patel et al., 2014; Russo and Marr, 2019). With the emergence of invasive and metastatic diseases and challenging clinical management caused by multi-drug resistance (MDR), people have gradually realized that Klebsiella pneumoniae with high virulence (HvKP) induces the infection even though MDR occurs based on hypervirulence (Karampatakis et al., 2023). This is a significant threat to patients and a difficult challenge to doctors.

HvKp carries multiple virulence factors, including capsule, lipopolysaccharide, and PLVKP-like virulence plasmid (rmpA, rmpA2, iutA, terW, and silS), resulting in high mortality in mice (Liu et al., 2020; Li et al., 2021). As the main virulence factor of Kp, capsules have been identified as various serotypes. Ten common serotypes belong to HvKp, including K1, K2, K5, K16, K54, K57, and so on, and these strains are characterized by their ability to produce capsular polysaccharide (CPS), supporting bacterial killing and elimination from the human immune system, such as complement and neutrophils (Hsu et al., 2016; Su et al., 2020). The predominant strains of HvKP are K1 and K2, with K57 following closely. Conversely, the occurrence of K54 is infrequently reported worldwide, especially in China. Furthermore, HvKP is usually hypervirulent. K1-Kp can delay the apoptosis of human neutrophils, and K2-Kp has a strong antiserum-killing ability in mouse models with LD₅₀ ≤ 10³ CFU (Lee et al., 2017; Wang et al., 2021). K57-Kp has a solid antiserum-killing ability (Wei et al., 2021). Turton’s study has indicated that the virulence of the K54-Kp strain ST29 is parallel to that of the ST86 strain, with high virulence belonging to K2-Kp (Turton et al., 2018). Previous studies on K54-Kp are only some case reports (Chuang et al., 2013; Iwasaki et al., 2017). Due to the limited number of cases, a comprehensive understanding of the epidemiology, clinical characteristics, and molecular and virulence features of K54-Kp remains elusive.

Furthermore, our initial research indicated that K54-Kp possesses a remarkable ability to form biofilms, piquing our interest in this study. Hence, through clinical data, drug susceptibility profiles, PCR amplification of virulence and resistance genes, and virulence phenotype experiments, we aim to systematically uncover the clinical and microbial attributes, molecular and virulence characteristics, and the specific molecular traits of this population.

Kp has attracted much attention recently due to its clinical invasion and drug resistance. It reportedly accounts for 86% of clinical infections, which has become the chief pathogen of healthcare-related infections (Guo et al., 2017; Liao et al., 2022). As a member of HvKP, K54-Kp should have some of the above-related characteristics. Therefore, we aimed to describe its molecular and clinical infection characteristics comprehensively. As expected, K54-Kp was not a dominant group, with a low prevalence rate, which was in line with the results of Liao’s (Liao et al., 2022). Our findings indicated that fewer strains were detected in 2016 and 2017. However, there was a gradual increase in 2018 and 2019, suggesting that the prevalence of K54-KP strains has been rising over the years. This trend indicates that they may become even more widespread in the future. Furthermore, while the main sources of the specimens in 2018 were from the neurosurgical ward and from the ICU ward in 2019, sputum remained the dominant specimen type. In 2018, the strains especially came from sputum in neurosurgery wards, and in 2019, they were primarily from ICU wards, where sputum was still the majority. K1/K2 is deemed to be closely related to community infections(Siu et al., 2012).

In contrast, our research found that K54-Kp was more from nosocomial infection (73.33%). Univariate analysis revealed that patients with hepatobiliary diseases were more susceptible to K54-KP infection. When patients with primary hepatobiliary disorders experience a high fever accompanied by liver pain, it is essential to consider the possibility of a liver abscess caused by K54-Kp as the primary diagnosis. Therefore, clinicians should be vigilant and monitor these patients closely.

The drug sensitivity results revealed a high resistance rate of K54-Kp to common clinical antibiotics. The trend of drug resistance increased significantly over the years, particularly during 2017-2018, and peaked in 2018-2019. Additionally, 66.67% of the strains in this study were classified as MDR-Kp, with CRKP accounting for more than half of these cases. The emergence of MDR-Kp with high drug resistance poses a significant challenge and makes K54-Kp the next “superbacterium,” leading to greater clinical drug resistance and more complicated management(Algammal et al., 2023). Moreover, morbidity and mortality rates are expected to rise considerably.

The string test is a widely used approach to identify strains with HM. In the present study, only 36.7% of strains were positive, while all of them contained the rmpA/rmpA2 gene, which could facilitate the production of CPS and perform a high-viscosity phenotype. However, certain strains that carried the mentioned genes did not exhibit high viscosity characteristics, indicating that solely relying on the string test results is insufficient to determine whether a strain is HvKP (Sheng et al., 2022). Additionally, genes such as iutA, terW, and silS also collectively contribute to the high-viscosity phenotype of HvKp (Li R. et al., 2020). The positive rate of the string test aligned with the results from the quantification of CPS, which is indicative of Kp virulence. When compared to NTUH-K2044, the CPS content of the K54-Kp isolate was found to be lower. This observation was unexpected for us, especially considering that wabG and wcaG, which code for lipopolysaccharide, had high prevalence rates (100% and 86.7%, respectively). These genes can jointly regulate capsule formation with rmpA (Zheng et al., 2018). Much like the rmpA mutant, capsule production is potentially linked to the CPS promoter. Furthermore, removing rmpA results in reduced promoter expression (Cheng et al., 2010), though the specific underlying mechanism requires further investigation.

MLST is a molecular typing tool used to trace the origin of bacteria owing to its good repeatability and high resolution. The well-known K1-Kp is ST23, K2-Kp mainly includes ST65 and ST86, and K57-Kp is ST412(Liu et al., 2021; Nakamura et al., 2021; Shao et al., 2021). At present, domestic and foreign literature reports on K54-Kp are mostly ST29. In our present study, ST29 was still dominant, mainly from blood specimens (7/11), which was rarely reported in the previous literature. Afterward, PFGE results showed that ST29 K54-Kp was concentrated in the B2 cluster, mainly distributed in neurosurgery in 2018, pointing out that there might be a clonal transmission of K5-Kp isolates in our hospital.

Numerous virulence genes, including pLVPK-like virulence genes (rmpA, rmpA2, iucA, iutA, iroN, etc.), occurred in K54-Kp strains. WabG, mrkD, aerobactin, and wcaG were the most, while the terW gene chiefly existed in ST29(p=0.015). Moreover, Alka Hasani has also found that the wcaG gene, associated with capsular polysaccharide synthesis, significantly correlates with K54-Kp isolates (p= 0.001) (Hasani et al., 2020). K54-Kp also took along diverse drug-resistance genes. The detection of drug-resistance genes agreed with the results of drug sensitivity, which sustains the argument that multiple drug-resistance genes jointly involved the drug-resistance characteristics of bacteria (Su et al., 2020). As a pivotal gene of carbapenem resistance, KPC enzymes were the most prevalent. Remarkably, we did not detect any K54 strains producing the OXA-48 gene. OXA-48, a class D carbapenemase enzyme, is regarded as the predominant carbapenemase gene in hvKp strains in Europe, whereas the KPC gene takes precedence in China (Liapis et al., 2014). The primary Kp strain carrying the OXA-48 gene is identified as the CG101 (ST 101) type in 38 out of 2,298 cases, as cited by Palmieri M et al. (Palmieri et al., 2020). The absence of the blaOXA-48 gene in our findings might be attributed to the geographical distribution trends of OXA-48 (Pitout et al., 2019). Furthermore, Kp strains producing the OXA-48 gene typically exhibit a lower resistance level to drugs compared to those with the KPC and ESBLs genes. However, in China, there’s a higher prevalence of resistance to meropenem and imipenem.

A notable observation was the coexistence of virulence and resistance genes. The hybridization data from S1-PFGE and southern blot revealed distinct molecular weights for the virulent and drug-resistant plasmids in the K54-Kp strains. We sought to discern if the plasmids containing both the rmpA2 and KPC genes were identical. Southern blot results indicated that while these strains housed heterozygous plasmids carrying both virulence and drug resistance genes, no fusion plasmid was present. According to Xie et al. (Xie et al., 2020), such heterozygous plasmids might encode both hazardous and resistant traits by amalgamating structural sections from two separate plasmids. Li et al. (Li D. et al., 2020) have detailed that two homologous regions encoding the mobile element shared by the plasmid and the Group II intron reverse transcriptase can recombine to form the Kp hybrid plasmid.

Biofilms serve as a formidable defense mechanism for sessile bacteria. Strains of K54-Kp involved in bloodstream infections are predisposed to biofilm formation, which results in enhanced virulence. Given that MDR-Kp strains can potentially form biofilms, we delved into their drug susceptibility profiles for deeper insights (James et al., 2019). Our findings revealed that while most strains were susceptible to carbapenem antibiotics, they demonstrated resistance to extended-spectrum β-lactam antibiotics. Notably, there was a significant propensity for ESBLs-Kp to form biofilms in bloodstream infections (P<0.05). The heightened biofilm-forming ability among K54-Kp strains derived from sources causing bloodstream infections not only facilitates the spread of resistance genes among bacteria but also augments the adaptability and survival of K54-Kp in hostile environments. In serum complement-mediated killing experiments, only 16.7% of the strains showed resistance to serum complement, all of which were resistant to carbapenem in sputum. In contrast to MDR bacteria, which typically have great adaptability or low virulence, carbapenem-sensitive strains display no resistance to complement-mediated death (Hennequin and Robin, 2016).

The virulence of ST29-K54Kp was lower than that of ST11 type and NTUH-K2044 in the Galleria mellonella model, contrary to Shao’s research(Shao C. et al., 2022). To further clarify, we analyzed further analysis the virulence genes carried by ST29-K54. The ST29-K54 was discovered to be more dominant in carrying the rmpA2, wcaG, and aerobactin genes (p<0.05), as opposed to the rmpA, iutA, and iroN genes. This finding could indicate that the strain’s gene-environment inhibited or hindered the expression of virulence genes, and the detailed mechanism requires further investigation. Additionally, in the Galleria mellonella model, the strains with positive biofilm development ability and serum resistance did not exhibit correspondingly high pathogenicity, which may also be associated with environmental factors of the strains, as well as the patient’s primary diseases, the number of complications, and the standard antibiotic treatment.

In conclusion, our study described the overall situation of K54-Kp in a Chinese-affiliated hospital. To our knowledge, this is the first systematic report of K54-Kp. K54-Kp showed a gradually increasing detection rate, highly invasive and pathogenic, and high biofilm formation ability of bloodstream infection strains. Furthermore, the prevalence of MDR-K54 Kp and the development of resistance mechanisms are triggering a global crisis. Therefore, supervision and attention should be strengthened in medical work. However, there are certain restrictions. To begin with, the quantity of samples should be enhanced so as to more extensively and completely depict the K54-KP characteristics. In addition, WGS sequencing need to be conducted with the aim to comprehend the strain’s genomic data when exploring the covert transmission of virulence plasmid in ST29-K54 kp. In-depth research should also be conducted on the mechanism of covert transmission of virulence plasmids, including whether or not it is connected to the expression of the genes contained by these plasmids (detection of plasmid copy number).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

JQ and DW performed the laboratory measurements and contributed equally to this manuscript. YL and NC made substantial contributions to the design and revised the article critically for important intellectual content. TX was in charge of organizing the supplementary experiment. JQ drafted the article. JM, QR, CW, BH participated in the analysis and interpretation of datas. LZ was responsible for the operation of the supplementary experiment. All authors read and approved the final article.