The pathogenesis of classical and hypervirulent Klebsiella pneumoniae strains vary significantly, particularly in their capsular structure, virulence genes, and iron acquisition strategies (Choby et al., 2020). Hypervirulent strains are distinguished by the production of K1 and K2 capsular types, with the mucoviscosity-associated gene (magA gene) unique to K1 strains. Sialic acid further enhances resistance to phagocytosis. Lipopolysaccharides (LPS) contribute to bacterial spread in the bloodstream and colonization of internal organs by providing resistance to serum killing through O-antigen and inhibiting phagocytosis via core polysaccharides.

Virulence is also increased by biofilm formation, mediated by Type 1 pili (T1P), which enhances phagocytosis through FimH, and Type 3 pili (T3P), which is implicated in lung infections (Choby et al., 2020). Iron acquisition plays a key role in virulence, with siderophores such as enterobactin, salmochelin, yersiniabactin, and aerobactin helping bacteria absorb host iron. While enterobactin is inhibited by the host molecule lipocalin-2, other siderophores evade this inhibition. Specific transporters like Ferric enterobactin receptor (FepA), salmochelin N (IroN), yersiniabactin Q (YbtQ), and Ferric aerobactin receptor (IutA) facilitate the uptake of trivalent iron in a siderophore-dependent manner, supporting bacterial survival in iron-limited conditions.Hypervirulent strains of Klebsiella pneumoniae carry key virulence factors encoded on a large virulence plasmid known as pLVPK. This plasmid includes the rmpA and rmpA2 genes, which regulate the mucoid phenotype, contributing to the hyperviscosity of these strains. Additionally, several iron acquisition systems are encoded on the same plasmid, enhancing the bacterium’s ability to acquire iron in the host environment, a crucial factor for its survival and virulence. The latest research shows that interpreting blaSHV alleles in Klebsiella pneumoniae genomes is complex due to various mutations, which can lead to extended-spectrum β-lactamase (ESBL) or β-lactamase inhibitor (BLI) resistance (Choby et al., 2020). The study systematically reviewed the experimental evidence for SHV enzyme function, reclassifying 37 SHV alleles and improving the mapping of genotype to phenotype. These findings have been integrated into the Kleborate v2.4.1 tool, enhancing the accuracy of gene classification and aiding in the understanding of antimicrobial resistance mechanisms.

The virulence factors of K. pneumoniae are encoded by the genes of the core genome and the accessory genome called chromosomal and plasmid-encoded genes (Wang et al., 2020). The virulence factors identified so far include capsular polysaccharides (CPS), lipopolysaccharide (LPS), siderophore, fimbriae, pilus, plasma membrane, and ribosomes (Wang et al., 2020). The hvKp-specific virulence plasmid and which of the defined virulence factors are located on this genomic element, which is primarily responsible for the hvKp pathotype. Figure 1 in the illustration demonstrates a side-by-side comparison of virulence factors in classical and highly virulent strains of K. pneumoniae. It’s worth noting that the siderophore is a feature shared with other pathogens like Enterobacter, Salmonella, and Yersinia. Understanding these distinctions in virulence factors is crucial for comprehending the varying levels of pathogenicity and clinical outcomes associated with different strains of K. pneumoniae. Cellular composition included capsule, nucleotide, plasmid, flagellum, pilus, plasma membrane, and ribosome. Figure 2 illustrates the immune response to K. pneumoniae infection. It provides a comprehensive view of the cellular composition involved in K. pneumoniae infection and the diverse strategies used for immunization, highlighting the key immune players in combating this pathogen.

The emergence of MDR K. pneumoniae is profoundly impacted by the improper use of broad-spectrum antibiotics, and the mechanisms of resistance exhibit great diversity. These mechanisms include hydrolytic modification of antibiotics, mutation of the antibiotic target site, reduced permeability, and overexpression of efflux pumps (Zhu et al., 2021). Modification of antibiotics by hydrolysis is a major mechanism of resistance, and K. pneumoniae can produce extended-spectrum β-lactamases (ESBLs) for the modification of available antibiotics (Zhu et al., 2021). β-lactam hydrolases target β-lactam antibiotics and extend their activity to mannose-binding lectin (MBL) and other carbapenemases. MBLs along with carbapenemases, can hydrolyze a wide range of β-lactam antibiotics, including penicillin, cephalosporins, aminoglutethimide, and carbapenems, leading to the ineffectiveness (Karampatakis et al., 2023; Figure 4). Another mechanism of resistance involves mutations in antibiotic target sites, particularly in the case of quinolones. Quinolone resistance arises from chromosomal mutations occurring in the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV. These mutations commonly occur in a localized domain of the GyrA and ParE subunits of gyrase and topoisomerase IV (Hooper and Jacoby, 2016).

Tigecycline sensitivity is reduced by chromosome-mediated modifications of the 30S and 16S ribosomal units, which are the targets of the tigecycline action (Jo and Ko, 2021). Additionally, mutations in plsC, rpsJ, trm, tet(A), and tet(M) genes also contribute to the decreased sensitivity to tigecycline (Sun et al., 2019). LPS modification systems facilitate the modification of LPS, resulting in reduced negative charge and decreased affinity for mucin, which ultimately leads to resistance (Linkevicius et al., 2013). Mutations in the two-component system PmrAB and PhoPQ, as well as their modulators (e.g., MgrB), can lead to the addition of L-Ara4N and PETN into lipid A within the LPS structure, consequently elevating the minimum inhibitory concentration (MIC) and decrease the susceptibility of microorganisms (Liu X. et al., 2022). Aminoglycoside resistance can arise from target site modifications caused by 16S rRNA methyltransferases (RMT), that modify specific nucleotide residues in the rRNA of the 16S subunit, preventing aminoglycosides from effectively binding to their intended target sites (Lv et al., 2020).

The reduction in permeability is related to the ineffectiveness of antibiotics in entering or crossing the bacterial outer membrane through pore protein channels to reach their sites of action (Vasan et al., 2022). Low intracellular concentration of antibiotics occurs when certain hydrophilic antibiotics, such as β-lactams, tetracyclines, chloramphenicol, and fluoroquinolones, diffuse through the outer membrane’s aqueous channels formed by pore proteins (Omp) (Richter et al., 2017). These porins proteins facilitate the passage of antibiotics, allowing them to traverse into the cell. The quantity, form, and quality of outer membrane pore proteins in various Gram-negative bacteria can be down-regulated or mutated, leading to reduced permeability and increased resistance (Vergalli et al., 2020). This effect may synergistically combine with basal or elevated expression of efflux transport proteins. Resistance to polymyxins in Salmonella enterica can be increased through the interaction of the periplasmic protein (YdeI) with the OmpD pore protein regulated by the two-component system of PhoPQ and PmrAB (Nang et al., 2019). Tigecycline resistance mechanism involves the downregulation of the outer membrane pore protein gene, ompK35, which leads to altered cell permeability, contributing to resistance (Park et al., 2023; Figure 4). The gene cluster tmexCD1-toprJ1 carried by plasmid, that encodes a resistant nodal superfamily RND-type efflux pump and responsible for conferring resistance to tetracyclines and also leads to reduced susceptibility to various clinically significant antimicrobial drugs (Lv et al., 2020). Overexpression of multidrug efflux systems belonging to the RND family of multidrug efflux pumps leads to reduced accumulation of antibiotics in the cytoplasm which provides bacteria with sufficient time to replicate and acquire resistance (Duraes et al., 2018). Tigecycline resistance in K. pneumoniae is associated with significant roles played by overexpression of the intrinsic chromosomally encoded RND-type efflux pumps AcrAB and OqxAB. These pumps are overexpressed due to mutations in transcriptional regulatory genes (ramR and acrR) (Lv et al., 2020). Additionally, the insertion of ISAba1 into the AdeS gene results in a truncated soluble AdeS kinase protein being produced through the promoter on ISAba1 which overexpress the AdeABC pump, resulting in tigecycline resistance (Jo and Ko, 2021; Figure 4).

There are three main mechanisms by which bacterial resistance genes disseminate among bacterial species; horizontal transfer of transposons, horizontal transfer of plasmids, and spread of clonal populations (Lv et al., 2020). The Tn3-based transposon Tn4401 is the most common mobile element containing bla_KPC. This transposon includes bla_KPC, Tn3 transposase gene (tnpA), Tn3 catabolic enzyme gene (tnpR), and two insertion sequences, Kpn6 and Kpn7 (Figure 4). Figure 4 illustrates the resistance mechanisms employed by K. pneumoniae. These mechanisms are critical for the bacterium’s survival and defense against antibiotics. The core structure in the horizontal transport of bla_KPC–2 may be involved in ISKpn27-bla_KPC–2-ISKpn6 (Zhang F. et al., 2021). The diverse 5bp target site sequence of Tn4401 supports its high transposon mobility. Figure 5 provides an overview of genetic mobile elements associated with the antibiotic resistance gene bla_KPC in K. pneumoniae. This gene is carried by two primary mobile elements: TN4401 and NTEKPC, which play a significant role in the spread of antibiotic resistance. A common hotspot for Tn4401 is transposon Tn1331, which gives rise to a heterozygous transposon structure (Ramirez et al., 2019). Tn1331 carries Tn3-like transposase and catabolic enzyme genes (tnpA and tnpR), aminoglycoside modifying enzyme genes aac(6′)-Ib and aadA1, as well as the β-lactamase genes bla_OXA–9 and bla_TEM–1. The bla_KPC gene was also found in non-Tn4401 mobile elements (NTE) (Figure 5). NTE_KPC is mainly present in non-ST258 K. pneumoniae or other non-K. pneumoniae species; whereas, bla_KPC in epidemic ST258 K. pneumoniae strains is exclusively carried on Tn4401.

Resistance genes can be transferred horizontally between bacteria via plasmids (Su et al., 2020). CRKP strains exhibit multi-drug resistance due to the presence of bla_KPC on large plasmids. These plasmids also carry other resistance determinants, conferring resistance to a range of antibiotics, including aminoglycosides, quinazolines, methicillin, sulfonamides, and tetracyclines (Ying et al., 2020). These resistance genes are highly transmissible, contributing to the dissemination of multi-drug resistance among CRKP strains. Mobile or transposable factors are consistently linked with qnr genes, especially ISCR1 and IS26. The qnr genes are typically found in multi-resistant plasmids alongside other resistance determinants. These plasmids encoded resistance by producing Qnr proteins, which shield target enzymes from the action of quinolones (Amereh et al., 2023). There are three known quinolone resistance mediators, namely Qnr proteins: QnrA, QnrB, and QnrS, which protect against quinolone inhibition leading to resistance to the target enzyme encoding DNA helicase and topoisomerase IV; whereas a variant of the aminoglycoside-modified acetyltransferase AAC(6′)-Ib acetylates certain quinolones, reduces antibiotic activity leading to resistance, and then completes dissemination by plasmid transfer into host cells (Hooper and Jacoby, 2015; Figure 6A). The carbapenemase gene transfer is linked with two IS26 elements of class I integrons. These elements are transferred at the interbacterial level by transmitting the resistance gene to the adapter conjugate (Ying et al., 2020). Gene bla_KPC is found on various types of plasmids (Figure 6B), and ColE-type plasmids, ranges in size from 10 to 300 kb (Chen et al., 2014). The genetic analysis of six bla_KPC-bearing plasmids from different incompatibility groups revealed a common structure among them. The Escherichia coli (E. coli) strains carrying bla_KPC, small Col-like plasmids are the second most prevalent type of plasmid and are commonly associated with KPC producers (Chen et al., 2014). These small vectors have been demonstrated to play a significant role in facilitating the interspecies spread of the qnr genes, which confers resistance to fluoroquinolones (Chen et al., 2014). Plasmid-mediated polymyxin/colistin resistance is attributed to the mcr family genes, which encode a group of phosphoethanolamine transferases responsible for facilitating the horizontal transfer of resistance to polymyxins (Wang et al., 2016; Wareth and Neubauer, 2021). These genes are found in different plasmids with diverse backbones and their size ranges from 58 to 251Kb (Gao et al., 2016). Common plasmids with mcr-1 can be used as helper plasmids to further promote plasmid propagation-mediated tigecycline resistance (Karki et al., 2021). These interconnected evolutionary fine-tunings may contribute to maintaining or enhancing the bacterial fitness of these prevalent clones and aid in the dissemination of clonal populations.

K. pneumoniae can elicit robust innate immune system activation and interact with neutrophils, macrophages, dendritic cells, and epithelial cells (Gonzalez-Ferrer et al., 2021). DC cells produce inflammatory cytokines, while macrophages are a major cellular target of IFN-γ (Van Elssen et al., 2010). NK cells are a major source of IL-22, and IL-17 to help in the clearance of Klebsiella by regulating the antimicrobial activity of the epithelium tissue. Collectively, these epithelium produces CXCL5 and Lipocalin 2, which in turn recruits neutrophils, and epithelial cells with the help of defensins and IL-8 (Ahn and Prince, 2020). The alveolar macrophage produces inflammatory cytokines IL-23 and type I IFN, and IL-23 which activates NK cells to produce cytotoxic effects (Wei et al., 2023; Figure 2C). K. pneumoniae can also induce adaptive immunity in humans, leading to the activation of germinal center B cells. These cells rapidly proliferate and undergo somatic hypermutation in their immunoglobulin variable genes. Follicular dendritic cells in vivo secrete T-cell signals CD40L and B-cell activating factor (Baff) that affect and regulate B-cell survival, differentiation, and B-cell production of antibodies that kill bacteria (Chen et al., 2019; Tang et al., 2021). The antigen-present cells recognize the antigen-presenting cell peptide and present it to a subset of T cells in a local lymph node (Roche and Furuta, 2015). The antigen-present cells release polarized cytokines (i.e., IL-23) that can activate Th17 and γδ T cells. These T cell subsets produce IL-17 and IL-22 and stimulate airway epithelial cells expression to mediate chemokines (for example, CXCL1)(Cai et al., 2010). Activated neutrophils engulf and kill bacteria, thereby enhancing K. pneumoniae clearance and local inflammation resolution (Figure 2D). To combat the infections caused by K. pneumoniae, a series of related vaccines have been developed, including whole-cell vaccine, capsular polysaccharide vaccine, LPS-related vaccine, protein vaccine, conjugate vaccine, ribosomal vaccine, reverse vaccine, DNA vaccine, and mRNA vaccine (Ranjbarian et al., 2023; Figure 2B).

Proteins are antigens and due to their ability to elicit an immune response, they can be used as vaccine candidates. The immunogenic proteins of K. pneumoniae are the external membrane proteins (EMPs) and pilin protein (Zhu et al., 2021). EMPs and type III pilus have been utilized in the development of experimental vaccines (Xie et al., 2020). Five EMPs have been used in a mice model and identified that these vaccines increase the production of antigen-specific IgG, IgG1, and IgG2a antibodies. In infection models of K. pneumoniae, it was observed that only Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 were capable of eliciting protective immune responses (Zhu et al., 2021). Furthermore, these protective effects were accompanied by distinct immune responses induced by the K. pneumoniae OmpA. Based on this, it can be inferred that Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 represent three promising candidate antigens that may trigger Th1, Th2, and Th17 immune responses. These antigens hold the potential to be developed into multivalent vaccines that are effective across different serotypes, offering protection against K. pneumoniae infections (Zhang B. Z. et al., 2021).

In a recent study, an integrated informatics and genome analysis pipeline was employed to rationally design a multiepitope subunit vaccine targeting four high-risk serotypes (Wang et al., 2022). The investigators initially conducted in silico epitope prediction and antigenicity screening, followed by molecular docking to characterize the interactions of the construct with Toll-like receptors (TLRs). Subsequently, molecular dynamics simulations were performed to evaluate the conformational stability of the vaccine in a simulated physiological fluid environment. This comprehensive computational framework enabled the identification of candidate epitopes with optimal immunogenicity, strong TLR binding affinities, and robust structural integrity under near physiological conditions.

Conjugate vaccines achieve long-lasting immune protective effects through a chemical process involving the covalent binding of polysaccharide antigens to carrier proteins, resulting in the formation of glycoproteins (Wantuch and Rosen, 2023). This covalent linkage enables the vaccine to elicit a stronger and more sustained immune response compared to using polysaccharide antigens alone. Among bacterial vaccines, glycoconjugate vaccines, which involve the binding of bacterial polysaccharides to carrier proteins, have emerged as one of the most successful approaches to date (Liu et al., 2016). The immunogenicity of polysaccharides is commonly enhanced by protein carrier conjugates. The conjugate vaccines, which utilized the pilin protein of K. pneumoniae and the O-specific polysaccharide (OPS) of E. coli serotype K12, exhibited significantly increased antibody titers against OPS semi-antigens and bacterial cilia as carrier proteins. Currently, a glycoconjugate vaccine is in development, comprising four K. pneumoniae OPS serotypes conjugated to Pseudomonas aeruginosa (P. aeruginosa) flagellin (Hegerle et al., 2018). The OPS vaccine has shown immunogenicity in animal models that received passively transferred antibodies induced by the OPS vaccine exhibited protection against systemic cKp infection. However, it is more suitable for targeting cKp strains due to the masking effect of the podoconjugate polysaccharide found in highly virulent isolates, which can obscure the OPS antigen. Concurrently, a nano-coupled vaccine has been developed utilizing the OPS with galactose repeats from K. pneumoniae (Peng et al., 2021). This OPS-based nanocouple vaccine shows promising potential as a potent antigen within a nanovaccine preparation platform (Liu et al., 2016). Additionally, a bioconjugation approach has been developed, by utilizing glycoengineered E. coli that expresses K1 and K2 antigens of K. pneumoniae under the modulation of rmpA. This innovative method triggers a serotype-specific IgG response in mice, effectively protecting them from infection caused by K1 and K2 strains of K. pneumoniae (Liu et al., 2016). The coupling of OPS with PA Fla and PA glycoconjugate vaccines leads to a formulation that generates antibody titers against four K. pneumoniae OPS types and two PA Fla antigens. This enhanced formulation can provide broad protection against multiple K. pneumoniae OPS types and P. aeruginosa flagellin (Liu et al., 2016). The development of conjugate vaccines encounters certain limitations, mainly attributed to the utilization of a single type of carrier protein and the high production costs involved, to restrict the flexibility and cost-effectiveness of the vaccine development process.

Reverse vaccinology involves the analysis of a pathogens genome sequencing to identify antigenic determinants. Subsequently, high-throughput cloning, expression, and purification techniques are applied to obtain recombinant proteins of the antigenic determinants. The candidate antigens are then subjected to in vivo and in vitro evaluations to screen for the protective antigens, which can be further utilized in vaccine development. This approach allows for more targeted and efficient screening of potential vaccine candidates. Compared to traditional vaccine research methods, reverse vaccines offer greater convenience, broader scope, and enhanced safety. Employed antigenomic techniques have been applied to screen 169 antigenic proteins of K. pneumoniae from clinical diagnoses (Lundberg et al., 2013). Subsequently, through in vitro analysis and murine infection models, eight novel proteins capable of inducing active immunity and providing protection were identified (Mehmood et al., 2020). This highlights the potential of reverse vaccinology in identifying promising vaccine candidates efficiently and effectively. These eight newly identified proteins are expected to be utilized in the preparation of K. pneumoniae vaccines. Reverse vaccinology paves the way for a novel approach to vaccine development, enabling the prediction and discovery of previously undiscovered immunogens. This approach is anticipated to play an increasingly significant role in future vaccine development endeavors.

In recent years, mRNA vaccine technology has emerged as a promising avenue in the fight against MDR K. pneumoniae infections. These innovative vaccines are tailored to target specific genes associated with drug resistance or the production of virulence factors in K. pneumoniae (Khalid and Poh, 2023).

The concept behind mRNA vaccines is ingenious. They are designed to encode precise segments of these critical genes, stimulating the immune system to mount a robust response against the encoded proteins. For example, mRNA vaccines can effectively zero in on β-lactamase genes, provoking an immune response that directly counters the β-lactamase enzyme’s activity. This, in turn, diminishes the enzymes ability to deactivate antibiotics, thereby enhancing the antibiotics effectiveness.

By harnessing the body immune response mechanisms, mRNA vaccines represent a highly promising strategy for addressing antibiotic-resistant bacteria. They hold immense potential in the prevention and control of MDR K. pneumoniae infections by precisely targeting drug resistance and virulence factors.

However, it’s crucial to emphasize that rigorous testing and validation in both preclinical and clinical studies are imperative to establish the safety and efficacy of these mRNA vaccines. These steps are critical in our collective efforts to combat antibiotic resistance effectively and manage MDR K. pneumoniae infections in a rapidly evolving medical landscape. Developing mRNA vaccines for K. pneumoniae presents several scientific challenges that need to be addressed for successful vaccine implementation. These challenges include:

Tackling these challenges requires collaboration between researchers, clinicians, and regulatory agencies. Continued advancements in mRNA vaccine technology and a deep understanding of K. pneumoniae biology and pathogenesis will be pivotal in developing effective vaccines against this MDR pathogen (Hegerle et al., 2018).

Vaccine development has undergone a shift from using whole-cell vaccines comprised of intact microorganisms expressing multiple antigens to subunit or non-cellular vaccines containing well-defined, purified antigens. This transition has resulted in reduced reactogenicity of the vaccine and enabled the production of repeatable and stable vaccines (Mba et al., 2023). Bacterially secreted outer membrane vesicle (OMV), known as extracellular vesicles (EVs), are the most widely used antimicrobial membrane nanomaterials. Bacterial bionanobacterial vesicle (BBV) vaccines offer a unique advantage with their dual function of inducing both bacterial-specific humoral and cellular immune responses. These vaccines have demonstrated the capability to enhance animal survival rates, reduce lung inflammation, and lower bacterial burden (Opoku-Temeng et al., 2019). The establishment of BBV vaccine platforms has greatly optimized the technology for developing vaccines against MDR K. pneumoniae (Hirabayashi et al., 2021). In addition, microencapsulated vaccines, multi-epitope vaccines, and DNA vaccines can elicit specific immune responses against K. pneumoniae (Assoni et al., 2021). Also, iron carriers and membrane receptor cells have been explored as potential vaccine candidates, which play a crucial role in the virulence of the pathogen and are uniquely positioned to facilitate rapid recognition by the host immune system (Gorden et al., 2018). Another vaccine candidate involves live attenuated strains of D-glutamate-deficient mutants, which lack a key structural component of the bacterial cell wall (Cabral et al., 2017). The vaccine effectively induces a protective immune response against systemic infections caused by various K. pneumoniae strains.