Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Bacterial extracellular vesicles affect endocrine therapy in MCF7 cells.

BACKGROUND: : The microbiome is important in the development and progression of breast cancer. This study investigated the effects of microbiome derived from Klebsiella on endocrine therapy of breast cancer using MCF7 cells. The bacterial extracellular vesicles (EVs) that affect endocrine therapy were established through experiments focused on tamoxifen efficacy. METHODS: : The microbiomes of breast cancer patients and healthy controls were analyzed using next-generation sequencing. Among microbiome, Klebsiella was selected as the experimental material for the effect on endocrine therapy in MCF7 cells. MCF7 cells were incubated with tamoxifen in the absence/presence of bacterial EVs derived from Klebsiella pneumoniae and analyzed by quantitative real-time polymerase chain reaction and Western blot. RESULTS: : Microbiome derived from Klebsiella is abundant in breast cancer patients especially luminal A subtype compared to healthy controls. The addition of EVs derived from K pneumoniae enhances the anti-hormonal effects of tamoxifen in MCF7 cells. The increased efficacy of tamoxifen is mediated via Cyclin E2 and p-ERK. CONCLUSION: : Based on experiments, the EVs derived from K pneumoniae are important in hormone therapy on MCF7 cells. This result provides new insight into breast cancer mechanisms and hormone therapy using Klebsiella found in the microbiome.

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae.

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Kpi, a chaperone-usher pili system associated with the worldwide-disseminated high-risk clone Klebsiella pneumoniae ST-15.

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.

The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae.

For ∼30 years, two distinct groups of clinical isolates of Klebsiella pneumoniae have been recognized. Classical strains (cKp) are typically isolated from patients with some degree of immunocompromise and are not virulent in mouse models of infection whereas hypervirulent strains (hvKp) are associated with community acquired invasive infections and are highly virulent in mouse models of infection. Hyperproduction of capsule and a hypermucoviscous colony phenotype have been strongly associated with the hypervirulence of hvKp strains. Recent studies have begun to elucidate the relationship between capsule gene expression, hypermucoviscosity and hypervirulence. Additionally, genes associated with hyperproduction of capsule and hypermucoviscosity in hvKp strains have been identified in a few cKp isolates. However, it is not clear how the acquisition of these genes impacts the virulence of cKp isolates. A better understanding of the potential risks of these strains is particularly important given that many of them are resistant to multiple antibiotics, including carbapenems.

Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles.

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

Lavender essential oil induces oxidative stress which modifies the bacterial membrane permeability of carbapenemase producing Klebsiella pneumoniae.

Misuse of antibiotics in the clinical and agricultural sectors has caused the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae which contributes a threat to human health. In this study, we assessed the feasibility of lavender essential oil (LVO) as an antimicrobial agent in combinatory therapy with meropenem in suppressing the growth of carbapenemase-producing K. pneumoniae (KPC-KP). Synergistic interactions between LVO and meropenem were detected, which significantly reduce the inhibitory concentration of both LVO and meropenem by 15 and 4-fold respectively. Comparative proteomic profiling identified a disruption in the bacterial membrane via oxidative stress that was indicated by loss of membrane and cytoplasmic proteins and the upregulation of oxidative regulators. As a proof of concept, zeta potential measurements showed a change in cell surface charge while outer membrane permeability measurement indicated an increase in membrane permeability following exposure to LVO. This was indicative of a disrupted outer membrane. Ethidium bromide influx/efflux assays demonstrated no significant efflux pump inhibition by LVO, and scanning electron microscopy revealed irregularities on the cell surface after exposure to LVO. Oxidative stress was also detected with increased level of ROS and lipid peroxidation in LVO-treated cells. In conclusion, our data suggest that LVO induced oxidative stress in K. pneumoniae which oxidizes the outer membrane, enabling the influx of generated ROS, LVO and meropenem into the bacterial cells, causing damage to the cells and eventually death.

Transcriptional regulation of galF by RcsAB affects capsular polysaccharide formation in Klebsiella pneumoniae NTUH-K2044.

RcsAB is an atypical two-component regulatory system that can regulate exopolysaccharide biosynthesis and is involved in the virulence of K. pneumoniae. The gene galF is well known as a gene involved in the biosynthesis of capsular polysaccharide (CPS). The specific DNA identification sequence for transcriptional regulation of RcsAB was found to be present in the promoter region of galF. This study aimed to detect the function of RcsAB in virulence and in biofilm and CPS formation. In addition, the transcriptional regulation of the galF gene in K. pneumoniae was studied. To determine the function of rcsAB gene, the wild-type K. pneumoniae strain NTUH-K2044 and the rcsAB knockout and complemented strains were used. The results showed decreased virulence, biofilm formation, and CPS levels in the rcsAB knockout strain. Complementation of the knockout by introducing an rcsAB fragment on an expression plasmid partially restored the virulence, biofilm, and CPS functions of the knockout strain. It indicated that the rcsAB genes might affect CPS formation and virulence of K. pneumonia. RT-qPCR, EMSA and DNase I footprinting assays were conducted to identify the transcriptional regulation of galF by RcsAB. RcsAB was seen to bind to the galF promoter-proximal region, and the binding site was further identified to be located from -177 bp to -152 bp upstream of the galF promoter. In conclusion, RcsAB could regulate the transcription of the galF gene positively by binding to the galF promoter DNA directly, and then affects the CPS formation of K. pneumonia.

Inactivating NADH:quinone oxidoreductases affects the growth and metabolism of Klebsiella pneumoniae.

NADH:quinone oxidoreductases (NQOs) act as the electron entry sites in bacterial respiration and oxidize intracellular NADH that is essential for the synthesis of numerous molecules. Klebsiella pneumoniae contains three NQOs (NDH-1, NDH-2, and NQR). The effects of inactivating these NQOs, separately and together, on cell metabolism were investigated under different culture conditions. Defective growth was evident in NDH-1-NDH-2 double and NDH-1-NDH-2-NQR triple deficient mutants, which was probably due to damage to the respiratory chain. The results also showed that K. pneumoniae can flexibly use NQOs to maintain normal growth in single NQO-deficient mutants. And more interestingly, under aerobic conditions, inactivating NDH-1 resulted in a high intracellular NADH:NAD+ ratio, which was proven to be beneficial for 2,3-butanediol production. Compared with the parent strain, 2,3-butanediol production by the NDH-1-deficient mutant was increased by 46% and 62% in glycerol- and glucose-based media, respectively. Thus, our findings provide a practical strategy for metabolic engineering of respiratory chains to promote the biosynthesis of 2,3-butanediol in K. pneumoniae.

A Far-Red Fluorescent DNA Binder for Interaction Studies of Live Multidrug-Resistant Pathogens and Host Cells.

Transgene expression of green fluorescent protein (GFP) has facilitated the spatiotemporal investigation of host-pathogen interactions; however, introduction of the GFP gene remains challenging in drug-resistant bacteria. Herein, we report a novel far-red fluorescent nucleic acid stain, 6-TramTO-3, which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered divergent interaction strategies of antibiotic-resistant and antibiotic-sensitive Klebsiella strains with immune cells. 6-TramTO-3 constitutes an off-the-shelf reagent for real-time analysis of bacterial infection, including strains for which the use of genetically encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug-resistant bacteria.

Controlled graphene encapsulation: a nanoscale shield for characterising single bacterial cells in liquid.

High-resolution single-cell imaging in their native or near-native state has received considerable interest for decades. In this research, we present an innovative approach that can be employed to study both morphological and nano-mechanical properties of hydrated single bacterial cells. The proposed strategy is to encapsulate wet cells with monolayer graphene with a newly developed water membrane approach, followed by imaging with both electron microscopy (EM) and atomic force microscopy (AFM). A computational framework was developed to provide additional insights, with the detailed nanoindentation process on graphene modelled based on the finite element method. The model was first validated by calibration with polymer materials of known properties, and the contribution of graphene was then studied and corrected to determine the actual moduli of the encapsulated hydrated sample. Application of the proposed approach was performed on hydrated bacterial cells (Klebsiella pneumoniae) to correlate the structural and mechanical information. EM and energy-dispersive x-ray spectroscopy imaging confirmed that the cells in their near-native stage can be studied inside the miniaturised environment enabled with graphene encapsulation. The actual moduli of the encapsulated hydrated cells were determined based on the developed computational model in parallel, with results comparable with those acquired with wet AFM. It is expected that the successful establishment of controlled graphene encapsulation offers a new route for probing liquid/live cells with scanning probe microscopy, as well as correlative imaging of hydrated samples for both biological and material sciences.

Automated patterning and probing with multiple nanoscale tools for single-cell analysis.

The nano-manipulation approach that combines Focused Ion Beam (FIB) milling and various imaging and probing techniques enables researchers to investigate the cellular structures in three dimensions. Such fusion approach, however, requires extensive effort on locating and examining randomly-distributed targets due to limited Field of View (FOV) when high magnification is desired. In the present study, we present the development that automates 'pattern and probe' particularly for single-cell analysis, achieved by computer aided tools including feature recognition and geometric planning algorithms. Scheduling of serial FOVs for imaging and probing of multiple cells was considered as a rectangle covering problem, and optimal or near-optimal solutions were obtained with the heuristics developed. FIB milling was then employed automatically followed by downstream analysis using Atomic Force Microscopy (AFM) to probe the cellular interior. Our strategy was applied to examine bacterial cells (Klebsiella pneumoniae) and achieved high efficiency with limited human interference. The developed algorithms can be easily adapted and integrated with different imaging platforms towards high-throughput imaging analysis of single cells.

Emergence of two distinct subpopulations from Klebsiella pneumoniae grown in the stimulated microgravity environment.

AIM: To isolate and characterize the two phenotypically distinct subpopulations from Klebsiella pneumoniae clonal cultures grown in the simulate microgravity environment. MATERIALS & METHODS: Here clonal culture of K. pneumoniae strain ATCC BAA-1705 was grown within a vertically rotating wall vessel bioreactor. Microscopic, colony staining, biofilm assays and quantitative proteomics were used to define the features of subpopulations. RESULTS: Two subpopulations were isolated based on colony appearance and bacterial morphology and indicated the different capability of biofilm formation and antibiotics resistance. CONCLUSION: These findings would raise a possibility of understanding the adaptive roles of bacterial subpopulations formed under certain conditions from the viewpoint of population variation.

Potent bactericidal activity of silver nanoparticles synthesized from Cassia fistula fruit.

We demonstrated one-step synthesis of silver nanoparticles (AgNPs) from Cassia fistula fruit extract and their antibacterial activity against E. coli and K. pneumoniae. Biogenic AgNPs were characterized by scanning electron microscopy, X-Ray diffraction and fourier transform infrared spectroscopy. Results confirmed spherical shaped AgNPs with an average crystallite size of ∼69 nm. Dose-dependent (0, 10, 20, 40 and 80 µg mL-1) growth kinetic studies showed 100% potency against E. coli (20 µg mL-1) and K. pneumoniae (80 µg mL-1) after 1 and 5 h, respectively. Surface morphology analysis revealed formation of groove/pits in the lysed cell membrane that eventually led to bacterial death.

Polymyxin B in combination with meropenem against carbapenemase-producing Klebsiella pneumoniae: pharmacodynamics and morphological changes.

Combination therapy provides a useful therapeutic approach to overcome resistance until new antibiotics become available. In this study, the pharmacodynamics, including the morphological effects, of polymyxin B (PMB) and meropenem alone and in combination against KPC-producing Klebsiella pneumoniae clinical isolates was examined. Ten clinical isolates were obtained from patients undergoing treatment for mediastinitis. KPCs were identified and MICs were measured using microbroth dilution. Time-kill studies were conducted over 24 h with PMB (0.5-16 mg/L) and meropenem (20-120 mg/L) alone or in combination against an initial inoculum of ca. 106 CFU/mL. Scanning electron microscopy (SEM) was employed to analyse changes in bacterial morphology after treatment, and the log change method was used to quantify the pharmacodynamic effect. All isolates harboured the blaKPC-2 gene and were resistant to meropenem (MICs ≥8 mg/L). Clinically relevant PMB concentrations (0.5, 1.0 and 2.0 mg/L) in combination with meropenem were synergistic against all isolates except BRKP28 (polymyxin- and meropenem-resistant, both MICs >128 mg/L). All PMB and meropenem concentrations in combination were bactericidal against polymyxin-susceptible isolates with meropenem MICs ≤16 mg/L. SEM revealed extensive morphological changes following treatment with PMB in combination with meropenem compared with the changes observed with each individual agent. Additionally, morphological changes decreased with increasing resistance profiles of the isolate, i.e. increasing meropenem MIC. These antimicrobial effects may not only be a summation of the effects due to each antibiotic but also a result of differential action that likely inhibits protective mechanisms in bacteria.

A nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule.

Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 µM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin; genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the divalent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.

[Effect of Klebsiella pneumoniae KbvR regulator on bacterial biofilm formation and capsular synthesis].

OBJECTIVE: To construct the KbvR gene of LuxR family deletion mutant and complementation strains from Klebsiella pneumoniae NTUH-K2044 and analyze the effect of KbvR on bacterial growth, biofilm formation and capsular synthesis. METHODS: A KbvR gene deletion mutant strain was constructed using the suicide vector pKO3-Km, and the gene fragment including KbvR coding region, promoter area and transcription termination area were amplified and cloned into pGEM-T-easy plasmid to construct KbvR complementation strain. The growth curves of the wild-type strain, KbvR gene deletion mutant strain and complementation strain were observed to assess the effect of KbvR on bacterial growth. Crystal violet staining method was used to measure the effect of KbvR on biofilm formation; the effect of KbvR on capsular synthesis was detected using string test, centrifugal test and RT-PCR. RESULTS: The KbvR deletion mutant and complementation strains were constructed successfully. KbvR gene did not affect the growth of the bacteria, but biofilm formation and capsular synthesis were attenuated in KbvR deletion mutant strain. CONCLUSION: As a transcription factor of the LuxR family orphans of the quorum sensing system, KbvR positively regulates bacterial biofilm formation by affecting capsular synthesis.

A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.

First evidence of polar flagella in Klebsiella pneumoniae isolated from a patient with neonatal sepsis.

The genus Klebsiella belongs to the family Enterobacteriaceae, and is currently considered to be non-motile and non-flagellated. In the present work, 25 Klebsiella strains isolated from nosocomial infections were assessed for motility under different growth conditions. One Klebsiella isolate, KpBUAP021, demonstrated a swim-like motility phenotype. The K. pneumoniae genotype was confirmed by 16S rRNA and rpoB gene sequence analysis. Multilocus sequence typing analysis also revealed that the KpBUAP021 strain places it in the ST345 sequence type, and belongs to the phylogenetic Kpl group. Transmission electron microscopy and the Ryu staining technique revealed that KpBUAP021 expresses polar flagella. Finally, the presence of fliC, fliA and flgH genes in this K. pneumoniae strain was confirmed. This report presents the first evidence for flagella-mediated motility in a K. pneumoniae clinical isolate, and represents an important finding related to its evolution and pathogenic potential.