Zebrafish Larvae Microinjection and Automated Fluorescence Microscopy for Studying Klebsiella pneumoniae Infection and the Host Immune Response.

Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

Mucosal sugars delineate pyrazine vs pyrazinone autoinducer signaling in Klebsiella oxytoca.

Virulent Klebsiella oxytoca strains are associated with gut and lung pathologies, yet our understanding of the molecular signals governing pathogenesis remains limited. Here, we characterized a family of K. oxytoca pyrazine and pyrazinone autoinducers and explored their roles in microbial and host signaling. We identified the human mucin capping sugar Neu5Ac as a selective elicitor of leupeptin, a protease inhibitor prevalent in clinical lung isolates of K. oxytoca, and leupeptin-derived pyrazinone biosynthesis. Additionally, we uncovered a separate pyrazine pathway, regulated by general carbohydrate metabolism, derived from a broadly conserved PLP-dependent enzyme. While both pyrazine and pyrazinone signaling induce iron acquisition responses, including enterobactin biosynthesis, pyrazinone signaling enhances yersiniabactin virulence factor production and selectively activates the proinflammatory human histamine receptor H4 (HRH4). Our findings suggest that the availability of specific carbohydrates delineates distinct autoinducer pathways in K. oxytoca that may have differential effects on bacterial virulence and host immune responses.

Bovine Neutrophil ß-Defensin-5 Provides Protection against Multidrug-Resistant Klebsiella pneumoniae via Regulating Pulmonary Inflammatory Response and Metabolic Response.

Klebsiella pneumoniae (K. pneumoniae), a kind of zoonotic bacteria, is among the most common antibiotic-resistant pathogens, and it causes nosocomial infections that pose a threat to public health. In this study, the roles of synthetic bovine neutrophil ß-defensin-5 (B5) in regulating inflammatory response and metabolic response against multidrug-resistant K. pneumoniae infection in a mouse model were investigated. Mice were administrated intranasally with 20 µg of B5 twice and challenged with K. pneumoniae three days after B5 pretreatment. Results showed that B5 failed to directly kill K. pneumoniae in vitro, but it provided effective protection against multidrug-resistant K. pneumoniae via decreasing the bacterial load in the lungs and spleen, and by alleviating K. pneumoniae-induced histopathological damage in the lungs. Furthermore, B5 significantly enhanced the mRNA expression of TNF-α, IL-1ß, Cxcl1, Cxcl5, Ccl17, and Ccl22 and obviously enhanced the rapid recruitment of macrophages and dendritic cells in the lungs in the early infection phase, but significantly down-regulated the levels of TNF-α, IL-1ß, and IL-17 in the lungs in the later infection phase. Moreover, RNA-seq results showed that K. pneumoniae infection activated signaling pathways related to natural killer cell-mediated cytotoxicity, IL-17 signaling pathway, inflammatory response, apoptosis, and necroptosis in the lungs, while B5 inhibited these signaling pathways. Additionally, K. pneumoniae challenge led to the suppression of glycerophospholipid metabolism, the phosphotransferase system, the activation of microbial metabolism in diverse environments, and metabolic pathways in the lungs. However, B5 significantly reversed these metabolic responses. Collectively, B5 can effectively regulate the inflammatory response caused by K. pneumoniae and offer protection against K. pneumoniae. B5 may be applied as an adjuvant to the existing antimicrobial therapy to control multidrug-resistant K. pneumoniae infection. Our study highlights the potential of B5 in enhancing pulmonary bacterial clearance and alleviating K. pneumoniae-caused inflammatory damage.

Deep Intraclonal Analysis for the Development of Vaccines against Drug-Resistant Klebsiella pneumoniae Lineages.

Despite its medical relevance, there is no commercial vaccine that protects the population at risk from multidrug-resistant (MDR) Klebsiella pneumoniae infections. The availability of massive omic data and novel algorithms may improve antigen selection to develop effective prophylactic strategies. Up to 133 exposed proteins in the core proteomes, between 516 and 8666 genome samples, of the six most relevant MDR clonal groups (CGs) carried conserved B-cell epitopes, suggesting minimized future evasion if utilized for vaccination. Antigens showed a range of epitopicity, functional constraints, and potential side effects. Eleven antigens, including three sugar porins, were represented in all MDR-CGs, constitutively expressed, and showed limited reactivity with gut microbiota. Some of these antigens had important interactomic interactions and may elicit adhesion-neutralizing antibodies. Synergistic bivalent to pentavalent combinations that address expression conditions, interactome location, virulence activities, and clone-specific proteins may overcome the limiting protection of univalent vaccines. The combination of five central antigens accounted for 41% of all non-redundant interacting partners of the antigen dataset. Specific antigen mixtures represented in a few or just one MDR-CG further reduced the chance of microbiota interference. Rational antigen selection schemes facilitate the design of high-coverage and "magic bullet" multivalent vaccines against recalcitrant K. pneumoniae lineages.

Topical adjunctive treatment with flagellin augments pulmonary neutrophil responses and reduces bacterial dissemination in multidrug-resistant K. pneumoniae infection.

Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice. Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers. Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses. Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR-K. pneumoniae, thereby reducing bacterial dissemination to distant organs.

Distinctive features and prognostic utility of neutrophil in severe patients with Klebsiella pneumoniae infection.

Background: Neutrophil plays a pivotal role in the management of Klebsiella pneumoniae infection. Delineate the clinical characteristics and prognostic utility of neutrophil in severe patients with K. pneumoniae infection are crucial for clinical management and prognostic assessment. Methods: K. pneumoniae patients with different infection sites were enrolled from Medical Information Mart for Intensive Care IV and eICU Collaborative Research Database. Temporal variations of neutrophil counts within 30 days of clinical onset were examined using locally weighted scatterplot smoothing curves. Logistic regression analysis was performed to assess the relationship between neutrophil counts and hospital mortality. Results: A total of 1,705 patients caused by K. pneumonia were included in the study. The non-survivor group exhibited a comparatively older age and a higher proportion of K. pneumoniae infections originating from respiratory and bloodstream sources compared to the survivor group (38.4% vs 21.1%, p<0.0001, and 15.1% vs 10.3%, p=0.021). Patients combined with multiple drug resistance strains, respiratory infection, liver disease, and above 60 years exhibited a specific dynamic process of neutrophil levels. Neutrophils counts peaked at admission and 1-2 weeks later. There was a 'U'-shaped relationship between neutrophil counts and hospital mortality. Conclusions: Neutrophils in K. pneumoniae infected patients have distinctive features and dynamic clinical trajectories. Close monitoring of severe patients infected with K. pneumoniae upon admission and during the first 1-2 weeks after admission is of utmost importance, particularly for patients with a neutrophil count exceeding 8.0×109/L.

Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization.

The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Oral administration of Lactobacillus plantarum expressing aCD11c modulates cellular immunity alleviating inflammatory injury due to Klebsiella pneumoniae infection.

BACKGROUND: Klebsiella pneumoniae (KP), responsible for acute lung injury (ALI) and inflammation of the gastrointestinal tract, is a zoonotic pathogen that poses a threat to livestock farming worldwide. Nevertheless, there is currently no validated vaccine to prevent KP infection. The development of mucosal vaccines against KP using Lactobacillus plantarum (L. plantarum) is an effective strategy. RESULTS: Firstly, the L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c were constructed via homologous recombination to express the aCD11c protein either inducibly or constitutively. Both NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c strains could enhance the adhesion and invasion of L. plantarum on bone marrow-derived dendritic cells (BMDCs), and stimulate the activation of BMDCs compared to the control strain NC8-pSIP409 in vitro. Following oral immunization of mice with NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c, the cellular, humoral, and mucosal immunity were significantly improved, as evidenced by the increased expression of CD4+ IL-4+ T cells in the spleen, IgG in serum, and secretory IgA (sIgA) in the intestinal lavage fluid (ILF). Furthermore, the protective effects of L. plantarum against inflammatory damage caused by KP infection were confirmed by assessing the bacterial loads in various tissues, lung wet/dry ratio (W/D), levels of inflammatory cytokines, and histological evaluation, which influenced T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood and lung. CONCLUSIONS: Both the inducible and constitutive L. plantarum strains NC8-pSIP409-aCD11c' and NC8-pLc23-aCD11c have been found to stimulate cellular and humoral immunity levels and alleviate the inflammatory response caused by KP infection. These findings have provided a basis for the development of a novel vaccine against KP.

Inhibition of porcine origin Klebsiella pneumoniae capsular polysaccharide and immune escape by BY3 compounded traditional Chinese medicine residue fermentation broth.

Klebsiella pneumoniae (K. pneumoniae) is a gram-negative conditionally pathogenic bacterium that causes disease primarily in immunocompromised individuals. Recently, highly virulent K. pneumoniae strains have caused severe disease in healthy individuals, posing significant challenges to global infection control. Capsular polysaccharide (CPS), a major virulence determinant of K. pneumoniae, protects the bacteria from being killed by the host immune system, suggesting an urgent need for the development of drugs to prevent or treat K. pneumoniae infections. In this study, BY3 compounded traditional Chinese medicine residue (TCMR) was carried out using Lactobacillus rhamnosus as a fermentation strain, and BY3 compounded TCMR fermentation broth (BY3 fermentation broth) was obtained. The transcription of K. pneumoniae CPS-related biosynthesis genes after treatment with BY3 fermentation broth was detected using quantitative real-time polymerase chain reaction. The effects of BY3 fermentation broth on K. pneumoniae serum killing, macrophage phagocytosis, complement deposition and human ß-defensin transcription were investigated. The therapeutic effect of BY3 fermentation broth on K. pneumoniae-infected mice was also observed, and the major active components of BY3 fermentation broth were analysed via LC‒MS analysis, network pharmacology, and molecular docking. The results showed that BY3 fermentation broth inhibited K. pneumoniae CPS production and downregulated transcription of CPS-related biosynthesis genes, which weakened bacterial resistance to serum killing and phagocytosis, while promoting bacterial surface complement C3 deposition and human ß-defensin expression. BY3 fermentation broth demonstrated safety and therapeutic effects in vivo and in vitro, restoring body weight and visceral indices, significantly reducing the organ bacterial load and serum cytokine levels, and alleviating pathological organ damage in mice. In addition, three natural compounds-oleanolic acid, quercetin, and palmitoleic acid-were identified as the major active components in the BY3 fermentation broth. Therefore, BY3 fermentation broth may be a promising strategy for the prevention or treatment of K. pneumoniae infections.

Computational strategies in Klebsiella pneumoniae vaccine design: navigating the landscape of in silico insights.

The emergence of multidrug-resistant Klebsiella pneumoniae poses a grave threat to global public health, necessitating urgent strategies for vaccine development. In this context, computational tools have emerged as indispensable assets, offering unprecedented insights into klebsiellal biology and facilitating the design of effective vaccines. Here, a review of the application of computational methods in the development of K. pneumoniae vaccines is presented, elucidating the transformative impact of in silico approaches. Through a systematic exploration of bioinformatics, structural biology, and immunoinformatics techniques, the complex landscape of K. pneumoniae pathogenesis and antigenicity was unravelled. Key insights into virulence factors, antigen discovery, and immune response mechanisms are discussed, highlighting the pivotal role of computational tools in accelerating vaccine development efforts. Advancements in epitope prediction, antigen selection, and vaccine design optimisation are examined, highlighting the potential of in silico approaches to update vaccine development pipelines. Furthermore, challenges and future directions in leveraging computational tools to combat K. pneumoniae are discussed, emphasizing the importance of multidisciplinary collaboration and data integration. This review provides a comprehensive overview of the current state of computational contributions to K. pneumoniae vaccine development, offering insights into innovative strategies for addressing this urgent global health challenge.

Vaccination with a trivalent Klebsiella pneumoniae vaccine confers protection in a murine model of pneumonia.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen and the major cause of healthcare-associated infections, which are increasingly complicated by the prevalence of highly invasive and hyper-virulent K. pneumoniae strains, necessitating the development of alternative strategies for combatting infections caused by this bacterium. In this study, we successfully constructed a fusion antigen called KP-Ag1, comprising three antigens (GlnH, FimA, and KPN_00466) that were previously identified through reverse vaccinology. Immunization with KP-Ag1 formulated with Al(OH)3 adjuvant elicited robust humoral and cellular immune response in mice, and conferred protective immunity in a murine model of K. pneumoniae lung infection. Further analysis of serum IgG subtypes from mice immunized with KP-Ag1 revealed a predominant IgG1 response, indicating that KP-Ag1 predominantly induces a Th2-biased immune response. Additionally, opsonophagocytic killing assay suggested that humoral immune responses play a pivotal role in mediating protection conferred by KP-Ag1. Moreover, KP-Ag1 was found to promote the activation and maturation of BMDCs in vitro, which is essential for subsequent efficient antigen presentation. More importantly, vaccination with KP-Ag1 demonstrated cross-protective efficacy against clinical isolates of K. pneumoniae varying in serotypes, antibiotic resistance, and virulence profiles. Therefore, KP-Ag1 holds promise as a candidate for K. pneumoniae vaccine development.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

Immunoprotective efficacy of 3 Klebsiella pneumoniae type I fimbriae proteins in a murine model.

Klebsiella pneumoniae is a primary cause of clinical mastitis in dairy cows, with prevention being crucial, as treatments often fail due to antimicrobial resistance. Recent studies identified type I fimbrial antigens of K. pneumoniae as promising vaccine candidates, but there are limited research data. In this study, 3 fimbriae genes (fimA, fimC and fimG) were cloned and recombinantly expressed in Escherichia coli and their protective efficacy against K. pneumoniae evaluated in a mouse model. All 3 recombinant fimbriae proteins elicited strong humoral immune responses in mice, significantly increasing IgG, IgG1 and IgG2a. Notably, using a model of mice challenged with an intraperitoneal injection of bacteria, FimG significantly reduced bacterial loads in the spleen and lung, whereas FimA and FimC had limited protection for these organs. Either active or passive immunization with FimG produced substantial protective effects in mice challenged with K. pneumoniae LD100; in contrast, the mortality rate in the FimA-immunized group was similar to that of the control group, whereas FimC had weak protection. We concluded that the FimG recombinant protein vaccine had a favorable protective effect, with potential for immunization against K. pneumoniae mastitis.

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

Exploring the journey: A comprehensive review of vaccine development against Klebsiella pneumoniae.

Klebsiella pneumoniae, a prominent nosocomial pathogen, poses a critical global health threat due to its multidrug-resistant (MDR) and hypervirulent strains. This comprehensive review focuses into the complex approaches undertaken in the development of vaccines against K. pneumoniae. Traditional methods, such as whole-cell and ribosomal-based vaccines, are compared with modern strategies, including DNA and mRNA vaccines, and extracellular vesicles (EVs), among others. Each method presents unique advantages and challenges, emphasising the complexity of developing an effective vaccine against this pathogen. Significant advancements in computational tools and artificial intelligence (AI) have revolutionised antigen identification and vaccine design, enhancing the precision and efficiency of developing multiepitope-based vaccines. The review also highlights the potential of glycomics and immunoinformatics in identifying key antigenic components and elucidating immune evasion mechanisms employed by K. pneumoniae. Despite progress, challenges remain in ensuring the safety, efficacy, and manufacturability of these vaccines. Notably, EVs demonstrate promise due to their intrinsic adjuvant properties and ability to elicit robust immune responses, although concerns regarding inflammation and antigen variability persist. This review provides a critical overview of the current landscape of K. pneumoniae vaccine development, stressing the need for continued innovation and interdisciplinary collaboration to address this pressing public health issue. The integration of advanced computational methods and AI holds the potential to accelerate the development of effective immunotherapies, paving the way for novel vaccines against MDR K. pneumoniae.

CircCDC42-encoded CDC42-165aa regulates macrophage pyroptosis in Klebsiella pneumoniae infection through Pyrin inflammasome activation.

The circular RNA (circRNA) family is a group of endogenous non-coding RNAs (ncRNAs) that have critical functions in multiple physiological and pathological processes, including inflammation, cancer, and cardiovascular diseases. However, their roles in regulating innate immune responses remain unclear. Here, we define Cell division cycle 42 (CDC42)-165aa, a protein encoded by circRNA circCDC42, which is overexpressed in Klebsiella pneumoniae (KP)-infected alveolar macrophages. High levels of CDC42-165aa induces the hyperactivation of Pyrin inflammasomes and aggravates alveolar macrophage pyroptosis, while the inhibition of CDC42-165aa reduces lung injury in mice after KP infection by inhibiting Pyrin inflammasome-mediated pyroptosis. Overall, these results demonstrate that CDC42-165aa stimulates Pyrin inflammasome by inhibiting CDC42 GTPase activation and provides a potential clinical target for pathogenic bacterial infection in clinical practice.

Dynamic immune status analysis of peripheral blood mononuclear cells in patients with Klebsiella pneumoniae bloodstream infection sepsis using single-cell RNA sequencing.

Background: Klebsiella pneumoniae is a common Gram-negative bacterium. Blood infection caused by K. pneumoniae is one of the most common causes of human sepsis, which seriously threatens the life of patients. The immune status of peripheral blood mononuclear cells (PBMCs) based on single-cell RNA sequencing (scRNA-seq) in acute stage and recovery stage of sepsis caused by K. pneumoniae bloodstream infection has not been studied. Methods: A total of 13 subjects were included in this study, 3 healthy controls, 7 patients with K. pneumoniae bloodstream infection in the acute stage (4 patients died), and 3 patients in the recovery stage. Peripheral blood of all patients was collected and PBMCs were isolated for scRNA-seq analysis. We studied the changes of PBMCs components, signaling pathways, differential genes, and cytokines in acute and recovery stages. Results: During K. pneumoniae acute infection we observed a decrease in the proportion of T cells, most probably due to apoptosis and the function of T cell subtypes was disorder. The proportion of monocytes increased in acute stage. Although genes related to their phagocytosis function were upregulated, their antigen presentation capacity-associated genes were downregulated. The expression of IL-1ß, IL-18, IFNGR1 and IFNGR2 genes was also increased in monocytes. The proportion of DCs was depleted during the acute stage and did not recover during sepsis recovery. DCs antigen presentation was weakened during the acute stage but recovered fast during the recovery stage. pDCs response to MCP-1 chemokine was weakened, they recovered it quickly during the recovery stage. B cells showed apoptosis both in the acute stage and recovery stage. Their response to complement was weakened, but their antigen presentation function was enhanced. The proportion of NK cells stable during all disease's stages, and the expression of IFN-γ gene was upregulated. Conclusion: The proportion of PBMCs and their immune functions undergo variations throughout the course of the disease, spanning from the acute stage to recovery. These findings provide new insights into the mechanism of PBMCs immune function during K. pneumoniae bloodstream infection sepsis and recovery and sets the basis for further understanding and treatment.

Lowering mortality risk in CR-HvKP infection in intestinal immunohistological and microbiota restoration.

Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40 %). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.

Characterization of mAbs against Klebsiella pneumoniae type 3 fimbriae isolated in a target-independent phage display campaign.

We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae. We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae. We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.