Klebsiella pneumoniae is a gram-negative and rod-shaped bacterium that belongs to the Enterobacteriaceae family (Adeolu et al., 2016), and was first described by Carl Friedlander in 1882. K. pneumoniae is considered a prominent nosocomial pathogen worldwide, and is a member of the “ESKAPE” (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens (Pendleton et al., 2013). General nosocomial infections caused by K. pneumoniae include pneumonia, bacteraemia, and urinary tract infections (UTIs) (Paczosa and Mecsas, 2016). The frequent use of antimicrobials has resulted in the development of carbapenem-resistant K. pneumoniae (CRKP) strains which first emerged in 1996 (Yigit et al., 2001). CRKP strains generally contain mobile genetic elements harbouring a variety of antimicrobial resistance genes, including beta-lactamase K. pneumoniae carbapenemase gene (bla _KPC), New Delhi metallo-β-lactamase gene (bla _NDM), and oxacillinase-48 gene (bla _OXA-48) (Lee et al., 2016; Zhang et al., 2015), among which bla _KPC has been found to be shared in approximately 78.6% (44/56) (Lin et al., 2020) and 89.5% (34/38) of isolates in two studies (Lin et al., 2018). CRKP strains account for over 30.0% of K. pneumoniae strains and present great challenges in clinical practice (Effah et al., 2020). CRKP is associated with mortality rates of 34.7% (17/49) for pneumonia, 37.8% (34/90) for bacteraemia, and 7.4% (9/121) for UTI (Hauck et al., 2016). Furthermore, CRKP treatment is associated with a higher medical cost than that of carbapenem-susceptible K. pneumoniae (Huang et al., 2018). CRKP constitutes a major public health issue, especially in endemic countries (Karampatakis et al., 2016). Therefore, CRKP control is considered a priority by the World Health Organization (World Health Organization, 2017). CRKP is usually denoted as classical K. pneumoniae (cKP) regarding its virulence (Russo and Marr, 2019; Zhang et al., 2020).

Hypervirulence in K. pneumoniae represents another major concern. Hypervirulent K. pneumoniae (HvKP) was first reported to cause pyogenic liver abscess (PLA) and septic endophthalmitis in seven healthy individuals (Liu et al., 1986). HvKP, which has a considerably lower median lethal dose (LD₅₀) than that of cKP in mouse model, generally produces various virulence factors such as hypercapsules, excessive siderophores, exopolysaccharides, and fimbriae (Paczosa and Mecsas, 2016; Russo and Marr, 2019). Apart from PLA, HvKP can also cause multiple invasive infectious diseases such as endogenous endophthalmitis, necrotising fasciitis, and meningitis, and the infection can undergo metastatic spread. PLA is endemic to East Asia and associated with a morbidity rate of 15.45 per 100,000 person-years in 2011 and a mortality rate of 8.2% (Chen et al., 2016; Siu et al., 2012). It has been estimated that 60% of endogenous endophthalmitis cases are associated with PLA caused by K. pneumoniae (Wong et al., 2000). Even with intravenous and intravitreal antimicrobial treatment, 89% of endophthalmitis cases show visual acuity of light perception or worse, and over 40% of affected eyes require evisceration or enucleation (Yang et al., 2007).

Recently, a combination of hypervirulence and extreme drug resistance has been reported in K. pneumoniae, thereby exacerbating the scarcity of effective treatments and resulting in high mortality (Zhang et al., 2015; Gu et al., 2018). The prevalence of infections caused by carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP) presents a global concern and a great challenge in clinical practice. However, the epidemiology of CR-HvKP and Hv-CRKP has not been extensively studied. The estimated prevalence of CR-HvKP/Hv-CRKP strains ranges among 5.0–15.0% among CRKP strains in mainland China, based on molecular determination or the Galleria mellonella (greater wax moth) lethality test (Zhang et al., 2020; Zhan et al., 2017). The gold standard method for evaluating the virulence of K. pneumoniae involves the use of mouse models, rather than the G. mellonella lethality test (Russo and MacDonald, 2020). Here, we analysed 436 clinical K. pneumoniae strains using molecular techniques and mouse lethality tests to elucidate the prevalence of CR-HvKP and Hv-CRKP strains.

A combination of carbapenem resistance and hypervirulence in õõõõõK. pneumoniae strains has been recently reported (Gu et al., 2018). However, the epidemiology of CR-HvKP and Hv-CRKP has not been extensively studied. To our knowledge, this is the first epidemiological surveillance study on bla _KPC(+)-HvKP and Hv-bla _KPC(+)-KP strains in China using a mouse lethality test to evaluate their prevalence.

In total, 13 key virulence genes in K. pneumoniae were investigated in this study; Figure 2 shows a remarkable divergence in their distribution. The extremely high detection rates of fimH, mrkD, entB, and wzi indicate ubiquitous production of type 1 fimbriae, type 2 fimbriae, enterobactins, and capsules. Another siderophore gene irp2 was present in a large proportion of the strains (76.1%), and is typically carried by ICEKp1 in the chromosome; The remaining 2 siderophore genes iucA and iroN showed detection rates of approximately 20.0–30.0%, and are usually harboured by pK2044 and pLVPK-like virulence plasmids (Struve et al., 2015). The virulence genes peg-344, p-rmpA, and p-rmpA2 are also present in pK2044- and pLVPK-like virulence plasmids (Russo and Marr, 2019), and therefore yielded similar detection rates to those of iucA and iroN (p > 0.05). The c-rmpA gene is present in the chromosome and often found in PLA specimens (Hsu et al., 2011). No such specimens were included in this study, which may explain the detection rate of 0.0%. The low detection rate of wzy-K1 (6.4%) suggests the rarity of K1 K. pneumoniae in clinical practice.

Various molecular factors were evaluated to screen for CR-HvKP and Hv-CRKP. Zhang et al. previously evaluated iucA, iroN, rmpA, and rmpA2 to check for the presence of virulence plasmids, and performed the G. mellonella lethality test to identify CR-HvKP, which yielded a rate of 5.2% (55/1052) for CR-HvKP (Zhang et al., 2020). We previously defined HvKP as: positive wzy-K1, ≥3 positive siderophore genes (entB, irp2, iroN, and iucA), or ≥1 positive capsule-regulating genes (p-rmpA2, c-rmpA/A2, and p-rmpA), and estimated a rate of 5.6% (29/521) for Hv-bla _KPC(+)-KP (Shon et al., 2013), which was also applied in this study. Harada et al. defined HvKP as strains carrying virulence genes, rmpA, rmpA2, iroBCDN, iucABCD, or iutA (Harada et al., 2019). Russo et al. confirmed that the G. mellonella lethality experiment cannot accurately differentiate HvKP from cKP (Russo and MacDonald, 2020). Thus, the mouse lethality test may represent the only approach to determine the exact prevalence of CR-HvKP/Hv-CRKP. In this study, only two bla _KPC(+)-HvKP strains, but no Hv-bla _KPC(+)-KP strains, were eventually confirmed using a mouse lethality test, showing a rate of 0.5% (2/436) for CR-HvKP which was far lower than that reported in other studies (Zhang et al., 2020; Hu et al., 2021). Owing to the predominance of KPC-induced carbapenem resistance (Lin et al., 2018), the actual prevalence of CR-HvKP should be approximately 0.5–1.0% in mainland China in 2017. The considerable difference in prevalence rates determined between this study and other reports highlights the need to elucidate why such biomarkers are not reliable and the difference between mouse and G. mellonella lethality tests. Zhang et al., reported that only one K. pneumoniae strain has been confirmed as CR-HvKP among three strains that harbour rmpA based on a mouse lethality test (Zhang et al., 2015). The fact that rmpA genes are non-functional in cKP may be attributed to different genetic backgrounds, although rmpA-related genes, such as kvrA, kvrB, and rcsB (Palacios et al., 2018; Walker et al., 2019), were found to be widely distributed in both cKP and HvKP (data not shown).

Although Hv-CRKP and CR-HvKP strains are currently emerging worldwide (Gu et al., 2018; Karlsson et al., 2019), our study revealed that the emergence of CR-HvKP is a relatively greater concern owing to its prevalence. In this study, two confirmed bla _KPC(+)-HvKP strains were found, including JS184 (K2) and TZ20 (K20), which showed no fitness cost, no hypercapsule production, and high expression of galF which is responsible for the synthesis of capsule precursor (Peng et al., 2018; Walker et al., 2019). However, JS184 showed a positive string test and exopolysaccharide production in contrast to TZ20. The reason for this is not known. In addition, JS184 and TZ20 also showed different ST and irp2 expression.

This study had a few limitations. First, only typical siderophore genes were referred to, but not their expression. Second, capsule staining is not sufficient to differentiate capsules of various thicknesses, which may impact virulence.

Taken together, our findings indicate that CR-HvKP may emerge more often than Hv-CRKP; the former accounted for less than 1.0% of the strains evaluated via mouse lethality tests among clinical K. pneumoniae strains in mainland China in 2017.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the School of Pharmacy, Fudan University (Shanghai, China).

DH, WC, and QZ contributed to conception of the study. ML, LY, ZY, YW, YH, GL, and XJ collected and identified the strains. DH, WC, QZ, PF, DT, and WW performed PCR and MLST analyses, string tests, capsular staining, periodic acid-Schiff staining, and fitness tests. PR and QM performed mouse lethality tests. DH, WC, and QZ wrote the manuscript which was revised by XJ and LY. All authors read and approved the final manuscript.

This study was supported by research grants from the National Natural Science Foundation of China (grants 81871692, 82172315, 82172315 and 81572031), Shanghai Municipal Key Clinical Specialty (Laboratory Medicine, No. shslczdzk03303), and the Shanghai Municipal Science and Technology Commission (grant number 19JC1413002).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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