Klebsiella pneumoniae (K. pneumoniae) is a common pathogen causing various nosocomial infections, including pneumonia, urinary tract infection, abdominal infection, and bacteremia. K. pneumoniae is now recognized as an urgent threat to human health, because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections (Shao et al., 2021a). A unique case of K. pneumoniae infection leading to liver abscess along with endophthalmitis was reported for the first time in Taiwan in the 1980s, and the causative organism was designated as hypervirulent K. pneumoniae (hvKp). Hence, hvKp has been recognized as another pathotype in addition to classical K. pneumoniae (cKp), associated with high degree of pathogenicity and mortality due to hypervirulence (Zhu et al., 2021). Currently, hvKp isolates have been reported mainly in Asia, Europe, and North America, and more recently in South America (Morales-León et al., 2021).

After invasive K. pneumoniae infections became a recognized clinical concern in Asia, several studies on the isolates reported the differences between cKp and hvKp. However, there is no specific feature that captures all hvKp strains to date (Russo and Marr, 2019). Clinical manifestations, capsule typing, the hypermucoviscous phenotype, and presence of virulence associated genes could be used to differentiate hvKp from cKp strains (Choby et al., 2020). The capsule polysaccharide of K. pneumoniae has been viewed as an important virulence factor that promotes resistance to phagocytosis and serum bactericidal activity. Currently, more than 100 capsular (K) serotypes of K. pneumoniae have been identified. Notably, many reports have shown that K1 and K2 serotypes are strongly associated with hvKp (Wyres et al., 2016). Nevertheless, capsular type K54, particularly representatives of sequence type (ST) 29, has also been associated with hypervirulence (Turton et al., 2018). In this study, we isolated a K54-ST29 K. pneumoniae from a patient with diabetes suffering multiple system infection in a Chinese Hospital. We characterized the genotypic and phenotypic features of the isolate.

The pathogen hvKp typically causes community-acquired pyogenic liver abscesses in patients with no clinical history (Arena et al., 2016). It is one of the leading cause of multisystem infection, involving pneumonia, hepatic and non-hepatic abscesses, endophthalmitis, meningitis, skin and soft tissue infections, and necrotizing fasciitis (Huang et al., 2018; Marr and Russo, 2019). Diabetes mellitus is considered as a significant risk factor for acquiring an hvKp infection, and this bacterial infection predominantly affects 55–60 year-old male individuals. In Taiwan and Singapore, hvKp was the most common cause of deep neck infections in patients with diabetes (Choby et al., 2020; Hirai et al., 2020). In the present case, the patient was a 58-year-old patient with diabetes, with similar clinical features reported previously. The clinical course of this patient revealed typical traits of hvKp infection (Eger et al., 2021). First, the infection was severe, due to the invasion of the pathogen into the bloodstream from liver abscesses. Second, multiple body sites were affected, including the respiratory and nervous systems, which is a hallmark of hvKp infection. The rapid disease progression and the severity of illness in this patient were remarkable.

So far, there are limited case reports on invasive infection caused by K54 hvKp. In order to further understand the evolutionary relationship of hvKp serotype K54, we downloaded the nucleotide sequences of eight K54 hvKp and analyzed their similarity with TAKPN-1. The results showed that seven hvKp were closely related to each other, suggesting parallel evolution of these isolates. A previous study showed that the most prevalent type found in K54 hvKp was ST29 (Turton et al., 2018). Chuang et al. (2013) reported a case of mycotic aneurysm caused by K.pneumoniae serotype K54 with ST29. Iwasaki et al. (2017) also reported a case of bacterial meningitis caused by hvKp capsular genotype K54 with ST 29. The latter case illustrated two significant clinical findings for cerebral focal lesion infected by K54 hvKp. First, it was agranuloma-like, similar to cryptococcoma and tuberculoma, during the subacute phase. Second, the lesion became undetectable during antibacterial therapy. Our case corroborated their clinical features. Unfortunately, we failed to isolate hvKp from CSF. The possible reasons include two aspects: first, the amount of hvKp in CSF was too small, and most bacteria invaded the brain parenchyma; second, antibiotics were used for 11 days before the CSF drainage. However, hvKp was detected in blood, liver abscess, and BALF. Therefore, it was considered that the brain lesion of this patient was caused by hvKp infection.

Except for capsule types, many other factors of hvKp attributed to its virulence and pathogenesis, including up to four siderophore systems for iron acquisition, increased capsule production, the type I and type III fimbriae, and the colibactin toxin (Wang et al., 2020). We compared the bacterial virulence factors of TAKPN-1 with that of K1 hvKp, K2 hvKp, and cKp. The results showed that three hvKp carried similar virulence factors. It is worth noting that four virulence factors exist only in TAKPN-1 but not in K1 or K2 hvKp. They are involved in iron acquisition and type VI secretion system. On the hand, 45 virulence factors only exist hvKp, which are involved in capsule production, iron acquisition and aerobactin, fimbriae related function, mucoid phenotype, and type VI secretion system. However, many virulence factors exist in both cKp and hvKp. It is possible that the different expression levels of virulence factors lead to the diversity of pathogenicity in cKps or hvKps, which needs to be further studied.

In the genome of TAKPN-1, 14 genes participated in fimbriae synthesis. Fimbriae allow bacteria to attach to the host cells to establish infection. A previous report confirmed observations that type III fimbriae contribute to biofilm formation and demonstrated that the expression of type III fimbriae is positively correlated with iron concentration (Shon et al., 2013). The invading pathogen, hvKp, encodes high affinity iron acquisition systems to counteract host nutritional immunity processes. HvKp expresses four siderophores for iron acquisition, and the accumulation of siderophore systems in hvKp suggests iron acquisition is a critical component of the emergence of this pathogen (Choby et al., 2020). Eleven genes related to iron transport and enterobactin synthesis were predicted in TAKPN-1. The definition of hvKp is controversial, however, several recent studies have reported that possessing iutA (aerobactin receptor-encoding gene), in addition to rmpA, is a characteristic hvKp trait (Hirai et al., 2020). Whole-genome sequencing showed the presence of iutA and absence of rmpA in TAKPN-1. Moreover, we did not find any plasmid in TAKPN-1. A previous study has also reported that some of the K54 K. pneumoniae of ST29 isolated from blood lack a virulence plasmid (Turton et al., 2018). We confirmed the high virulence of three clinical isolates by mouse toxicity test, which is a standard test for hvKp infection.

Although initial isolates of hvKp were antimicrobial sensitive, management challenges included rapid initiation of therapy to prevent subsequent spread, detection of occult abscess to enable source control, and appropriate site-specific therapy (for example, meningitis, endophthalmitis, and prostatic abscess; Russo and Marr, 2019). Additional imaging (including CT or MRI) may be helpful and required to recognize the infection and identify certain occult sites of infection, because site-specific antimicrobial regimens that achieve adequate drug concentrations are needed for an optimal outcome (McHardy et al., 2021). In the present case, three hvKp isolates were susceptible to all detected antibiotics, and meropenem was used because of multiple site infections, including the nervous system. This patient gradually recovered via antimicrobial therapy, and the abscesses shrank until disappeared. He was discharged from hospital after 55 days of antimicrobial therapy.

In summary, our study described the phenotypic and molecular features of K54-ST29 hvKp causing multi-system infection in a patient with type 2 diabetes. Our study raises the concern of K54 hvKp plausibly becoming a serious health threat, considering its growing prevalence. Further epidemiological studies are warranted to elucidate the virulence factors for K54 hvKp strains, pathophysiology, and clinical features associated with their infection.

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 studies involving human participants were reviewed and approved by the Ethics Committee of Taiwan City Central Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Taiwan City Central Hospital.

MJ and HW designed the experiments and revised the manuscript. CS carried out the experiments and wrote the manuscript. LX analyzed the data. PM contributed to experiment conception. All authors contributed to the article and approved the submitted version.

This study was supported by the National Natural Science Foundation of China (No. 81401696) and the Shandong Provincial Natural Science Foundation of China (No. ZR2016HL44).

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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The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.872140/full#supplementary-material