As reported previously, the MDR K. pneumoniae causes infections in patients with underlying diseases and is considered as cKP with high resistance rate but hypovirulence [2, 12, 14]. However, K. pneumoniae isolates from KP-PLA has converged hypervirulence and high antibiotic resistance, which limit the clinical treatment options . To date, little is known about the virulence characteristics of PLA-causing MDR strains. Therefore, 12 MDR K. pneumoniae strains were collected from 163 KP-PLA cases, and the virulence characteristics and molecular epidemiology were analyzed. To the best of our knowledge, this is the first study to analyze the virulence characteristics of the PLA-causing MDR strains.
Numerous studies have reported that antibiotic resistance rates are low in KP-PLA [8, 11, 12]. Moreover, the MDR strains were rare, and the patients infected were more likely accompanied by hepatobiliary diseases compared to patients infected with non-MDR strains (Table S1). Importantly, the uncontrollable infections and ineffective prognosis in patients with hepatobiliary diseases might be associated with recurrent bacteremia due to MDR bacteria. This phenomenon suggested that these MDR isolates might not be related to traditional cKP, and acquisition of MDR might not compromise the overall virulence, requiring further verification. However, the actual virulence of these MDR strains has not yet been well-evaluated.
The growth ability results suggestesd no fitness cost regarding the strains with resistant phenotype. In addition, hypermucoviscosity was considered as a surrogate marker of hvKP . In this study, the percentage of hypermucoviscous MDR strains was found to be slightly lower than that of typical hypervirulent strains. However, hypermucoviscosity might not be the only indicator of hypervirulence, wherein the polysaccharide capsule can protect K. pneumoniae from phagocytosis by immune cells and complement-mediated bactericidal action, which acts as a major virulence characteristic for hvKP . The results of capsular quantification revealed that the capsular content of PLA-causing MDR strains was higher than that of the standard strain and lower than that of the typical hypervirulent strains. The standard strain ATCC 700603 that recognized as classic K. pneumoniae is known for producing extended-spectrum β-lactamase (ESBL) enzymes that can hydrolyze oxyimino-β-lactams, resulting in resistance to these drugs, and its virulence is less than the typical hypervirulent K. pneumoniae . The data of capsular quantification was consistent with the data of the string test. Although the MDR strains and typical hypervirulent strains were sensitive to serum, the antiserum killing ability of these PLA-causing strains was significantly higher than that of the typical hypervirulent strains, which might be related to the content of capsular polysaccharide. Furthermore, the bacteria attaches to the surface of the host during the infectious process and are coated with polymers such as extracellular polysaccharides and DNA to form biofilms. The physical barrier formed by biofilms protect the bacteria from phagocytes and enzymes, improving the bacterial defenses against the host and antimicrobial resistance. This finding indicated that the biofilm formation ability of MDR strains was significantly higher than that of typical hypervirulent strains, which might be one of the reasons for the MDR strains to exhibit resistant phenotype. Moreover, G. mellonella larvae, acts as a model of invertebrate host infection, and has been used to explore the virulence and pathogenicity of K. pneumoniae strains . Although the siderophore genes were different in these PLA-causing MDR strains and hypervirulent strains, the virulence was assessed in terms of lethality, thereby suggesting that the siderophore transport systems need to be investigated further to clarify the correlation between siderophore utilization and bacterial virulence. The consistency between the clinical data and the results of phenotypic assays supported the theory that the PLA-causing MDR K. pneumoniae strains are hypervirulent.
The analysis of virulence genotypes also validated our hypothesis. K. pneumoniae strains are presented as 78 capsular serotypes, among which K1 and K2 are related to hvKP, and are strongly pathogenic to humans [29, 32]. In the present study, K1 or K2 serotypes accounted for half of the PLA-causing MDR strains, while all the typical hypervirulent strains belonged to K1 or K2 serotypes. Although K1 or K2 serotypes can regulate the virulence of K. pneumoniae, hypervirulence is not unique to these capsular serotypes . In addition, rmpA and aerobactin are vital genes for hypervirulence . rmpA regulates the synthesis of extracellular polysaccharide capsule to enhance the virulence [34–35], and aerobactin is essential for the growth and virulence of K. pneumoniae by regulating iron supply . In the present study, the prevalence of rmpA and aerobactin in the MDR strains was slightly lower than that of typical hypervirulent strains, indicating that the PLA-causing MDR strains are combined with hypervirulence. Importantly, wcaG, magA, and uge genes related to capsule synthesis are also prevalent in PLA-causing MDR strains [24, 36]. The inconsistency between the results of the capsule-related genes and hypermucoviscosity suggested that hypermucoviscosity is not the optimal factor for assessing hypervirulence, which should be assessed in conjunction with genotypes, other phenotypes, and clinical characteristics. Moreover, the high prevalence of siderophore genes, such as ybtA, entB, and kfuBC in the PLA-causing MDR strains suggested that the ability of iron uptake might be equivalent to that of typical hypervirulent strains. Furthermore, almost all the PLA-causing MDR strains carried fimH (related to type 1 fimbriae), mrkD (related to type 3 fimbriae), and ureA (an α-subunit of the urease, associated with invasion) [24, 37] and genetically corroborated with the virulence phenotype results. The results of these genes and adherence or invasion in biofilm formation in MDR strains might explain the enhanced resistance of PLA-causing MDR strains to antibiotics compared to the PLA-causing typical hypervirulent strains. Therefore, clinicians should focus on the MDR strains and select appropriate management strategies to treat KP-PLA to reduce bacterial adhesion and colonization.
MLST analysis uncovered the molecular epidemiology of PLA-causing MDR strains. The clones of these MDR strains were diverse and scattered, while the clones of typical hypervirulent strains almost belonged to ST23, as described previously . ST11-type K. pneumoniae is resistant to carbapenems, but not hypervirulent. However, the new ST11-type strain that has emerged in recent years is simultaneously hypervirulent, multidrug resistant, and transmissible, which could pose a serious threat to public health [9, 15]. Previous studies demonstrated that ST11 carbapenem-resistant hypervirulent strains are not found in KP-PLA. To the best of our knowledge, this is the first study to describe that one ST11 carbapenem-resistant strain might be MDR-hypervirulent K. pneumoniae and has been described in KP-PLA. Nonetheless, further surveillance and implementation are needed to control the dissemination of infection in hospital settings and community.