Employment of the Phage Cocktail as a Species-Specific Recognition Agent for Wide-Spectrum Detection of Bacterial Strains.

Phages have already been employed to detect bacteria because of their specific recognition capability and strong infectious activity toward their host. However, the reported single-phage-based techniques are inevitably restricted by false negative results that arose from extremely high strain specificity of phages. In this study, a cocktail composed of three Klebsiella pneumoniae (K. pneumoniae) phages was prepared as a recognition agent to broaden the recognition spectrum for detecting this bacterial species. A total of 155 clinically isolated strains of K. pneumoniae collected from four hospitals were adopted to test its recognition spectrum. A superior recognition rate of 91.6% for the strains was achieved due to the complementarity of the recognition spectra of the three phages composed of the cocktail. However, the recognition rate is as low as 42.3-62.2% if a single phage is employed. Based on the wide-spectrum recognition capability of the phage cocktail, a fluorescence resonance energy transfer method was established for detecting K. pneumoniae strains by employing fluorescein isothiocyanate labeled to the phage cocktail and Au nanoparticles labeled to p-mercaptophenylboronic acid as energy donors and acceptors, respectively. The detection process can be completed within 35 min, with a wide dynamic range of 5.0 × 102-1.0 × 107 CFU/mL. The application potential was verified by applying it to quantitate K. pneumoniae in different sample matrixes. This pioneer work opens an avenue for achieving wide-spectrum detection of different strains belonging to the same bacterial species with the phage cocktail.

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.

Discrimination between Carbapenem-Resistant and Carbapenem-Sensitive Klebsiella pneumoniae Strains through Computational Analysis of Surface-Enhanced Raman Spectra: a Pilot Study.

In clinical settings, rapid and accurate diagnosis of antibiotic resistance is essential for the efficient treatment of bacterial infections. Conventional methods for antibiotic resistance testing are time consuming, while molecular methods such as PCR-based testing might not accurately reflect phenotypic resistance. Thus, fast and accurate methods for the analysis of bacterial antibiotic resistance are in high demand for clinical applications. In this pilot study, we isolated 7 carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains and 8 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from clinical samples. Surface-enhanced Raman spectroscopy (SERS) as a label-free and noninvasive method was employed for discriminating CSKP strains from CRKP strains through computational analysis. Eight supervised machine learning algorithms were applied for sample analysis. According to the results, all supervised machine learning methods could successfully predict carbapenem sensitivity and resistance in K. pneumoniae, with a convolutional neural network (CNN) algorithm on top of all other methods. Taken together, this pilot study confirmed the application potentials of surface-enhanced Raman spectroscopy in fast and accurate discrimination of Klebsiella pneumoniae strains with different antibiotic resistance profiles. IMPORTANCE With the low-cost, label-free, and nondestructive features, Raman spectroscopy is becoming an attractive technique with great potential to discriminate bacterial infections. In this pilot study, we analyzed surfaced-enhanced Raman spectroscopy (SERS) spectra via supervised machine learning algorithms, through which we confirmed the application potentials of the SERS technique in rapid and accurate discrimination of Klebsiella pneumoniae strains with different antibiotic resistance profiles.

Modeling microbial ethanol production by S. aureus, K. pneumoniae, and E. faecalis under aerobic/anaerobic conditions - applicability to laboratory cultures and real postmortem cases.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse's colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols' concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae's models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.

Cryo-EM structures of LptB2FG and LptB2FGC from Klebsiella pneumoniae in complex with lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most Gram-negative bacteria. LPS transport from the inner membrane (IM) to the OM is achieved by seven lipopolysaccharide transport proteins (LptA-G). LptB2FG, an type VI ATP-binding cassette (ABC) transporter, forms a stable complex with LptC, extracts LPS from the IM and powers LPS transport to the OM. Here we report the cryo-EM structures of LptB2FG and LptB2FGC from Klebsiella pneumoniae in complex with LPS. The KpLptB2FG-LPS structure provides detailed interactions between LPS and the transporter, while the KpLptB2FGC-LPS structure may represent an intermediate state that the transmembrane helix of LptC has not been fully inserted into the transmembrane domains of LptB2FG.

Distinguishing Galactoside Isomers with Mass Spectrometry and Gas-Phase Infrared Spectroscopy.

Sequencing glycans is demanding due to their structural diversity. Compared to mammalian glycans, bacterial glycans pose a steeper challenge because they are constructed from a larger pool of monosaccharide building blocks, including pyranose and furanose isomers. Though mammalian glycans incorporate only the pyranose form of galactose (Galp), many pathogens, including Mycobacterium tuberculosis and Klebsiella pneumoniae, contain galactofuranose (Galf) residues in their cell envelope. Thus, glycan sequencing would benefit from methods to distinguish between pyranose and furanose isomers of different anomeric configurations. We used infrared multiple photon dissociation (IRMPD) spectroscopy with mass spectrometry (MS-IR) to differentiate between pyranose- and furanose-linked galactose residues. These targets pose a challenge for MS-IR because the saccharides lack basic groups, and galactofuranose residues are highly flexible. We postulated cationic groups that could complex through hydrogen bonding would offer a solution. Here, we present the first MS-IR analysis of hexose ammonium adducts. We compared their IR fingerprints with those of lithium adducts. We determined the diagnostic MS-IR signatures of the α- and ß-anomers of galactose in furanose and pyranose forms. We also showed these signatures could be applied to disaccharides to assign galactose ring size. Our findings highlight the utility of MS-IR for analyzing the unique substructures that occur in bacterial glycans.

Development of Drug-Resistant Klebsiella pneumoniae Vaccine via Novel Vesicle Production Technology.

Drug resistance of Klebsiella pneumoniae severely threatens human health. Overcoming the mechanisms of K. pneumoniae resistance to develop novel vaccines against drug-resistant K. pneumoniae is highly desired. Here, we report a technology platform that uses high pressure to drive drug-resistant K. pneumoniae to pass through a gap, inducing the formation of stable artificial bacterial biomimetic vesicles (BBVs). These BBVs had little to no bacterial intracellular protein or nucleic acid and had high yields. BBVs were efficiently taken up by dendritic cells to stimulate their maturation. BBVs as K. pneumoniae vaccines had the dual functions of inducing bacteria-specific humoral and cellular immune responses to increase animals' survival rate and reduce pulmonary inflammation and bacterial loads. We believe that BBVs are new-generation technology for bacterial vesicle preparation. Establishment of this BBV vaccine platform can maximally expand preparation technology for vaccines against drug-resistant K. pneumoniae.

The mechanism of action of the SSB interactome reveals it is the first OB-fold family of genome guardians in prokaryotes.

The single-stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism in bacteria. This protein performs two distinct, but closely intertwined and indispensable functions in the cell. SSB binds to single-stranded DNA (ssDNA) and at least 20 partner proteins resulting in their regulation. These partners comprise a family of genome guardians known as the SSB interactome. Essential to interactome regulation is the linker/OB-fold network of interactions. This network of interactions forms when one or more PXXP motifs in the linker of SSB bind to an OB-fold in a partner, with interactome members involved in competitive binding between the linker and ssDNA to their OB-fold. Consequently, when linker-binding occurs to an OB-fold in an interactome partner, proteins are loaded onto the DNA. When linker/OB-fold interactions occur between SSB tetramers, cooperative ssDNA-binding results, producing a multi-tetrameric complex that rapidly protects the ssDNA. Within this SSB-ssDNA complex, there is an extensive and dynamic network of linker/OB-fold interactions that involves multiple tetramers bound contiguously along the ssDNA lattice. The dynamic behavior of these tetramers which includes binding mode changes, sliding as well as DNA wrapping/unwrapping events, are likely coupled to the formation and disruption of linker/OB-fold interactions. This behavior is essential to facilitating downstream DNA processing events. As OB-folds are critical to the essence of the linker/OB-fold network of interactions, and they are found in multiple interactome partners, the SSB interactome is classified as the first family of prokaryotic, oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians.

Local interactions with the Glu166 base and the conformation of an active site loop play key roles in carbapenem hydrolysis by the KPC-2 ß-lactamase.

The Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a common source of antibiotic resistance in Gram-negative bacterial infections. KPC-2 is a class A ß-lactamase that exhibits a broad substrate profile and hydrolyzes most ß-lactam antibiotics including carbapenems owing to rapid deacylation of the covalent acyl-enzyme intermediate. However, the features that allow KPC-2 to deacylate substrates more rapidly than non-carbapenemase enzymes are not clear. The active-site residues in KPC-2 are largely conserved in sequence and structure compared with non-carbapenemases, suggesting that subtle alterations may collectively facilitate hydrolysis of carbapenems. We utilized a nonbiased genetic approach to identify mutants deficient in carbapenem hydrolysis but competent for ampicillin hydrolysis. Subsequent pre-steady-state enzyme kinetics analyses showed that the substitutions slow the rate of deacylation of carbapenems. Structure determination via X-ray diffraction indicated that a F72Y mutant forms a hydrogen bond between the tyrosine hydroxyl group and Glu166, which may lower basicity and impair the activation of the catalytic water for deacylation, whereas several mutants impact the structure of the Q214-R220 active site loop. A T215P substitution lowers the deacylation rate and drastically alters the conformation of the loop, thereby disrupting interactions between the enzyme and the carbapenem acyl-enzyme intermediate. Thus, the environment of the Glu166 general base and the precise placement and conformational stability of the Q214-R220 loop are critical for efficient deacylation of carbapenems by the KPC-2 enzyme. Therefore, the design of carbapenem antibiotics that interact with Glu166 or alter the Q214-R220 loop conformation may disrupt enzyme function and overcome resistance.

Variety of size and form of GRM2 bacterial microcompartment particles.

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi-permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production.

BACKGROUND: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. RESULTS: Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. CONCLUSIONS: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

Development of novel isatin-nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing-bacteria.

Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.

Structural and functional characterization of peptidyl-tRNA hydrolase from Klebsiella pneumoniae.

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2' or 3' hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 Å resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded ß-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.

Separation and Purification of Biogenic 1,3-Propanediol from Fermented Glycerol through Flocculation and Strong Acidic Ion-Exchange Resin.

In the present work, was investigated the separation and purification procedure of the biogenic 1,3-propanediol (1,3-PD), which is a well-known valuable compound in terms of bio-based plastic materials development. The biogenic 1,3-PD was obtained as a major metabolite through the glycerol fermentation by Klebsiella pneumoniae DSMZ 2026 and was subjected to separation and purification processes. A strong acidic ion exchange resin in H+ form was used for 1,3-PD purification from the aqueous solution previously obtained by broth flocculation. The eluent volume was investigated considering the removal of the secondary metabolites such as organic acids (acetic, citric, lactic, and succinic acids) and 2,3-butanediol (2,3-BD), and unconsumed glycerol. It was observed that a volume of 84 mL of ethanol 75% loaded with a flow rate of 7 mL/min completely remove the secondary metabolites from 10 mL of concentrated fermented broth, and pure biogenic 1,3-PD was recovered in 128 mL of the eluent.

Flocculation Efficiency and Mechanism of Carbamazepine by Microbial Flocculant Extracted from Klebsiella pneumoniae J1.

The microbial flocculant (MFX) extracted from Klebsiella pneumoniae J1 was used to remove carbamazepine in prepared wastewater and domestic sewage. The influence factors and flocculation mechanism were studied. The optimal carbamazepine removal conditions for MFX were pH of 7-8, 7 mL of flocculant, 0.1 mL of coagulant, and 35°C, and the removal rate reached 81.75%. MFX was efficient in the removal of carbamazepine in both domestic sewage (75.03%) and secondary sedimentation tank effluent (69.76%). The pseudo-first-order kinetic equation fitted the adsorption process better than the pseudo-second-order kinetic equation, which suggested that the adsorption was not pure chemical adsorption. The analysis of floc size suggested that the repulsive force between carbamazepine and MFX was weakened under alkalescent conditions, which can help the growth and coherence of flocs and increase the carbamazepine removal efficiency. Enough dosage of MFX can generate larger flocs, but excessive dosage of MFX will decrease the carbamazepine removal rate because of increase in electrostatic repulsion. The analysis of 3D-EEM and FTIR suggested that hydroxyl, amino, and carboxyl in MFX played an important role in the removal of carbamazepine. As an eco-friendly and highly efficient microbial flocculant, MFX has potential for practical applications in carbamazepine removal.

Structural and Functional Study of the Klebsiella pneumoniae VapBC Toxin-Antitoxin System, Including the Development of an Inhibitor That Activates VapC.

Klebsiella pneumoniae is one of the most critical opportunistic pathogens. TA systems are promising drug targets because they are related to the survival of bacterial pathogens. However, structural information on TA systems in K. pneumoniae remains lacking; therefore, it is necessary to explore this information for the development of antibacterial agents. Here, we present the first crystal structure of the VapBC complex from K. pneumoniae at a resolution of 2.00 Å. We determined the toxin inhibitory mechanism of the VapB antitoxin through an Mg2+ switch, in which Mg2+ is displaced by R79 of VapB. This inhibitory mechanism of the active site is a novel finding and the first to be identified in a bacterial TA system. Furthermore, inhibitors, including peptides and small molecules, that activate the VapC toxin were discovered and investigated. These inhibitors can act as antimicrobial agents by disrupting the VapBC complex and activating VapC. Our comprehensive investigation of the K. pneumoniae VapBC system will help elucidate an unsolved conundrum in VapBC systems and develop potential antimicrobial agents.

A fast impedance-based antimicrobial susceptibility test.

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach.

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe++ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.

Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant Klebsiella pneumoniae.

Conjugative pili are important in mediating bacterial conjugation and horizontal gene transfer. Since plasmid transfer can include antibiotic-resistance genes, conjugation is an important mechanism in the spread of antibiotic resistance. Filamentous bacteriophages have been shown to exist in two different structural classes: those with a 5-fold rotational symmetry and those with a one-start helix with approximately 5 subunits per turn. Structures for the F and the F-like pED208 conjugation pilus have shown that they have 5-fold rotational symmetry. Here, we report the cryoelectron-microscopic structure of conjugative pili from carbapenem-resistant Klebsiella pneumoniae, encoded on the IncFIIK pKpQIL plasmid, at 3.9 Å resolution and show that it has a one-start helix. These results establish that conjugation pili can exist in at least two structural classes, consistent with other results showing that relatively small perturbations are needed to change the helical symmetry of polymers.

Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions.

Klebsiella pneumoniae strain KPB-1 was isolated in early 2011 from the pleural fluid of an inpatient admitted at an Italian hospital. It was characterized to produce the KPC-3 carbapenemase and to belong to sequence type 512, a derivative of sequence type 258 clade II characterized by the cps-2 gene cluster. The K-antigen of K. pneumoniae KPB-1 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives and 1D and 2D NMR spectroscopy of the native polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KPB-1 capsular polysaccharide: The reactions catalysed by each glycosyltransferase in the cps-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.