Klebsiella MALDI TypeR: a web-based tool for Klebsiella identification based on MALDI-TOF mass spectrometry.

Klebsiella pathogens affect human and animal health and are widely distributed in the environment. Among these, the Klebsiella pneumoniae species complex, which includes seven phylogroups, is an important cause of community and hospital infections. The Klebsiella oxytoca species complex also causes hospital infections and antibiotic-associated haemorrhagic colitis. The unsuitability of currently used clinical microbiology methods to distinguish species within each of these species complexes leads to high rates of misidentifications that are masking the true clinical significance and potential epidemiological specificities of individual species. We developed a web-based tool, Klebsiella MALDI TypeR, a platform-independent and user-friendly application that enables uploading MALDI-TOF mass spectrometry data in order to identify Klebsiella isolates at the species complex and phylogroup levels. The tool, available at https://maldityper.pasteur.fr/, leverages a database of previously identified biomarkers that are specific for species complexes, individual phylogroups, or related phylogroups. We obtained 84%-100% identification accuracy depending on phylogroup. Identification results are obtained in a few seconds from batches of uploaded spectral data. Klebsiella MALDI TypeR enables fast and reliable identification of Klebsiella strains that are often misidentified with standard microbiological methods. This web-based identification tool may be extended in the future to other human bacterial pathogens.

Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning.

Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.

Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins.

Gram-negative bacteria belonging to the genus Klebsiella are important nosocomial pathogens, readily acquiring resistance to all known antibiotics. Bacteriocins, non-antibiotic antibacterial proteins, have been earlier proposed as potential therapeutic agents for control of other Gram-negative species such as Escherichia, Pseudomonas and Salmonella. This study is the first report describing pore-forming and peptidoglycan-degrading bacteriocins klebicins from Klebsiella. We have identified, cloned, expressed in plants and characterized nine pore-forming and peptidoglycan-degrading bacteriocins from different Klebsiella species. We demonstrate that klebicins can be used for broad and efficient control of 101 of the 107 clinical isolates representing five Klebsiella species, including multi-drug resistant pathovars and pathovars resistant to carbapenem antibiotics.

Aflatoxin B1 removal by three bacterial strains and optimization of fermentation process parameters.

Aflatoxin B1 (AFB1 ) removing bacterial strains were isolated from different habitats that were easily contaminated by AFB1 . Furthermore, the composition of the fermentation medium and conditions of fermentation process were optimized, including carbon source, nitrogen source, metal ions, temperature, initial pH value, inoculation volume, and culture broth volume. Using coumarin as the sole carbon and energy source, we primarily screened 31 strains, and 10 strains were found to be capable to remove AFB1 . Among them, the highest removal rate of 71.91% appeared in those isolated from rotten wood (poplar). Strains XY1, XY3, and T6 were carried out to identify, and the results were Klebsiella sp., Klebsiella pneumonia, and Pantoea sp., respectively. Corn cob powder and tryptone can significantly increase the AFB1 removal activity of these strains. The AFB1 removal activity of Klebsiella sp.XY1 and K. pneumonia XY3 can be enhanced by Ca2+ , and those of Pantoea sp. T6 can be enhanced by Cu2+ . Temperature and initial pH were positively correlated with the AFB1 removal activity of these strains in a certain range. This study not only provides reference for the screening and application of AFB1 removing bacteria, but also provides a basis for possible application in the food and feed industry.

Three-dimensional graphene for electrochemical detection of Cadmium in Klebsiella michiganensis to study the influence of Cadmium uptake in rice plant.

Cadmium (Cd), as a hazardous pollutant present in the environment as well as within biological samples, needs to be detected and remediated at the same time. Although many types of Cd detection techniques have been developed globally, there is no evidence to analyse Cd2+ ion electrochemically using graphene-based electrode for bioaccumulation of Cd in bacteria and plants. The present study describes the fabrication and characterization of a three-dimensional reduced graphene oxide-based electrode to detect bioaccumulation of Cd within the bacterial cell and rice tissues applying differential pulse voltammetry (DPV) technique. In addition, X-ray fluorescence (XRF) and X-ray diffraction (XRD) studies were performed as supporting tools for this study in the selected Cd resistant plant growth promoting rhizobacterial (PGPR) strain, Klebsiella michiganensis MCC3089. This strain was characterized based on its plant growth promoting (PGP) traits and exhibited bioaccumulation of Cd both under high and low Cd concentrations, of which the latter is more environmentally significant. The Cd-sequestration ability of this strain was found to reduce Cd uptake within rice seedlings.

Harvesting energy from cellulose through Geobacter sulfurreducens in Unique ternary culture.

In this study, both electricity generation capability and biodegradation process of carboxymethyl cellulose (CMC) were investigated using a defined ternary culture of Paenibacillus sp., Klebsiella sp. and Geobacter sulfurreducens as biocatalysts in MFCs. The maximum current density achieved by the ternary culture from CMC was 621 ±â€¯23 µA cm-2 in half-cell experiments and the maximum power density reached to 1146 ±â€¯28 mW m-2 in two-chamber MFCs. Meanwhile, the ternary culture also possessed three times higher CMC degradation capability compared to the pure strain J1. Besides, the key metabolite products, including cellobiose, glucose, acetate, were quantified by high performance liquid chromatography (HPLC) to illustrate the biodegradation process of CMC. The high electricity generation performance mainly resulted from the "division-of-labor" based cooperation and the enhanced extracellular electron transfer caused by the electron shuttle secreted by Klebsiella sp. This study highlighted the synergistic effect of specific community on electricity generation using CMC as carbon source, and laid the foundation for further optimization of more efficient and stable microbial consortia for bioenergy applications.

Typing and Species Identification of Clinical Klebsiella Isolates by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae, K. variicola, and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS.

Phosphate solubilization and chromium (VI) remediation potential of Klebsiella sp. strain CPSB4 isolated from the chromium contaminated agricultural soil.

In this study, an effort was made to identify an efficient phosphate solubilizing bacterial strain from chromium contaminated agricultural soils. Based on the formation of a solubilized halo around the colonies on Pikovskaya's agar amended with chromium (VI), 10 strains were initially screened out. Out of 10, strain CPSB4, which showed significantly high solubilization zone at different chromium concentrations, was selected for further study. The strain CPSB4 showed significant plant growth promotion traits with chromium (VI) stress under in-vitro conditions in broth. The plant growth promotion activities of the strain decreased regularly, but were not completely lost with the increase in concentration of chromium up to 200 mg L-1. On subjected to FT-IR analysis, the presence of the functional group, indicating the organic acid aiding in phosphate solubilization was identified. At an optimal temperature of 30 °C and pH 7.0, the strain showed around 93% chromium (VI) reduction under in-vitro conditions in broth study. In soil condition, the maximum chromium (VI) reduction obtained was 95% under in-vitro conditions. The strain CPSB4 was identified as Klebsiella sp. on the basis of morphological, biochemical and 16S rRNA gene sequencing. This study shows that the diverse role of the bacterial strain CPSB4 would be useful in the chromium contaminated soil as a good bioremediation and plant growth promoting agent as well.

Multiple Structures Disclose the Secretins' Secrets.

Bacterial secretins are outer membrane proteins that provide a path for secreted proteins to access the cell exterior/surface. They are one of the core components of secretion machines and are found in type II and type III secretion systems (T2SS and T3SS, respectively). The secretins comprise giant ring-shaped homo-oligomers whose precise atomic organization was only recently deciphered thanks to spectacular developments in cryo-electron microscopy (cryo-EM) imaging techniques.

Enhanced bioremoval of lead by earthworm-Lumbricus terrestris co-cultivated with bacteria-Klebsiella variicola.

Lead is a toxic heavy metal having devastating effects on the environment. The current study was focussed on bioremoval of lead using earthworm and lead resistant bacteria. Earthworms were subjected to various concentrations of lead in the soil bioaugmented with lead resistant bacteria (VITMVCJ1) to enhance the uptake of lead from the contaminated soil. Significant increase was observed in the length and body weight of the earthworms supplemented with lead resistant bacteria. Similarly, there was a substantial increase in the locomotion rate of the earthworms treated with lead resistant bacteria in comparison with the control. The gut micro flora of bacterial treated earthworms had increased number of bacterial cells than the untreated earthworms. The histopathological studies revealed the toxic effects of lead on the gut of earthworms indicating severe damage in lead resistant bacteria untreated worms, whereas the cells were intact in lead resistant bacteria treated worms. COMET assay showed increased DNA damage with higher tail DNA percent in the untreated earthworms. Further, the colonisation of the bacteria supplemented, onto the gut region of earthworms was observed by scanning electron microscopy. Atomic absorption spectrophotometry indicated a fair 50% uptake of lead within the biomass of earthworm treated with lead resistant bacteria.

Novel nano-particulated exopolysaccharide produced by Klebsiella sp. PHRC1.001.

In recent decades, microbial synthesis of polysaccharides with special functional properties has attracted increasing attention. This work reported a novel exopolysaccharide (EPS)-producing strain, Klebsiella sp. PHRC1.001 isolated from activated sludge. Physicochemical, rheological, emulsifying and toxicological properties of the obtained EPS were characterized. The EPS was mainly composed of d-glucose and l-arabinose, and was found to exist in aqueous solution in a nano-particulated form (∼50nm in diameter) with a strong tendency of aggregation. Rheological analysis showed that the EPS aqueous solution was a typical pseudoplastic fluid at higher concentration and could form weak gel upon alkaline treatment followed by neutralization. The EPS exhibited excellent emulsifying properties in stabilizing oil-in-water emulsions presumably by a Pickering mechanism owing to its nanoparticle structure. Acute toxicity test showed that 1.8g EPS per kg of body weight caused no toxic effect on mice. PHRC1.001 EPS has the potential to be a novel industrial polysaccharide.

The starch-binding domain family CBM41-An in silico analysis of evolutionary relationships.

Within the CAZy database, there are 81 carbohydrate-binding module (CBM) families. A CBM represents a non-catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch-binding domains from the family CBM41 that are usually part of pullulanases from the α-amylase family GH13. Currently there are more than 1,600 sequences classified in the family CBM41, almost exclusively from Bacteria, and so a study was undertaken in an effort to divide the members into relevant groups (subfamilies) and also to contribute to the evolutionary picture of family CBM41. The CBM41 members adopt a ß-sandwich fold (∼100 residues) with one carbohydrate-binding site formed by the side-chains of three aromatic residues that interact with carbohydrate. The family CBM41 can be divided into two basic subdivisions, distinguished from each other by a characteristic sequence pattern or motif of the three essential aromatics as follows: (i) "W-W-∼10aa-W" (the so-called Streptococcus/Klebsiella-type); and (ii) "W-W-∼30aa-W" (Thermotoga-type). Based on our bioinformatics analysis it is clear that the first and second positions of the motif can be occupied by aromatic residues (Phe, Tyr, His) other than tryptophan, resulting in the existence of six different carbohydrate-binding CBM41 groups, that reflect mostly differences in taxonomy, but which should retain the ability to bind an α-glucan. In addition, three more groups have been proposed that, although lacking the crucial aromatic motif, could possibly employ other residues from remaining parts of their sequence for binding carbohydrate. Proteins 2017; 85:1480-1492. © 2017 Wiley Periodicals, Inc.

Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.

Biosorption of Pb (II) from aqueous solution by extracellular polymeric substances extracted from Klebsiella sp. J1: Adsorption behavior and mechanism assessment.

The adsorption performance and mechanism of extracellular polymeric substances (EPS) extracted from Klebsiella sp. J1 for soluble Pb (II) were investigated. The maximum biosorption capacity of EPS for Pb (II) was found to be 99.5 mg g(-1) at pH 6.0 and EPS concentration of 0.2 g/L. The data for adsorption process satisfactorily fitted to both Langmuir isotherm and pseudo-second order kinetic model. The mean free energy E and activation energy Ea were determined at 8.22- 8.98 kJ mol(-1) and 42.46 kJ mol(-1), respectively. The liquid-film diffusion step might be the rate-limiting step. The thermodynamic parameters (ΔG(o), ΔH(o) and ΔS(o)) revealed that the adsorption process was spontaneous and exothermic under natural conditions. The interactions between EPS system and Pb (II) ions were investigated by qualitative analysis methods (i.e Zeta potential, FT-IR and EDAX). Based on the strong experimental evidence from the mass balance of the related elements participating in the sorption process, an ion exchange process was identified quantitatively as the major mechanism responsible for Pb (II) adsorption by EPS. Molar equivalents of both K(+) and Mg(2+) could be exchanged with Pb(2+) molar equivalents in the process and the contribution rate of ion exchange to adsorption accounted for 85.72% (Δmequiv = -0.000541).

Competitive adsorption of heavy metals by extracellular polymeric substances extracted from Klebsiella sp. J1.

The adsorption of Cu(2+) and Zn(2+) by extracellular polymeric substances (EPS) extracted from Klebsiella sp. J1 and competitive adsorption mechanism were investigated. Equilibrium adsorption capacities of Cu(2+) (1.77mMg(-1)) on Klebsiella sp. J1 EPS were higher than those of Zn(2+) (1.36mMg(-1)) in single systems. The competitive Langmuir and Langmuir-Freundlich isotherm models were proven to be effective in describing the experimental data of binary component system. The three dimensional sorption surfaces of binary component system demonstrated that the presence of Cu(2+) more significantly decreased the sorption of Zn(2+), but the sorption of Cu(2+) was not disturbed by the presence of Zn(2+). FTIR and EEM results revealed the adsorption sites of Cu(2+) entirely overlapped with those of Zn(2+). Cu(2+) and Zn(2+) showed competitive adsorption in binary systems, and Cu(2+) was preferentially adsorbed because of the stronger complexation ability of the protein-like substances in Klebsiella sp. J1 EPS.

[Changes of endotoxin concentration in blood serum in patients with uncomplicated acute myocardial Q-infarction].

The effectiveness of acute myocardial infarction (AMI) treatment does not correspond to high material costs for the study of its pathogenesis and development of new drugs. This circumstance gives the grounds to assume existence of nowadays unknown mechanisms of emergence and development of this disease. High probability of participation of endotoxin (ET) in the pathogenesis of AMI was theoretically proved by us for more than a quarter of the century ago, but it's clinical evidence to date is not found yet. As a result of the study a significant increase of endotoxin (ET) concentration in the blood serum of patients with AMI increasing from 1 to 14 day of the disease has been found. In women the concentration of ET was higher than in men. It allows to qualify the EA as a factor probably influencing the known difference in AMI tolerance in men and women. The source of ET were Bacteroides (most often--67.8% of patients), Klebsiella, Pseudomonas, Proteus, Escherichia coli. One or two bacteria more often took part in the development of EA. In 9.1% of patients the etiology of EA could not be verified, what indicates the presence of other sources of EA, not evaluated in this study. In 25% of patients with AMI serologic evidence of systemic candidiasis, caused by candida Albicans, has been found, what is able to enhance the biological effects of ET.

Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection.

Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 µg/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).

Modification of chimeric (2S, 3S)-butanediol dehydrogenase based on structural information.

A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 Å. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.

Signal peptide-independent secretory expression and characterization of pullulanase from a newly isolated Klebsiella variicola SHN-1 in Escherichia coli.

A strain with the power to produce extracellular pullulanase was obtained from the sample taken from a flour mill. By sequencing its 16S rDNA, the isolate was identified as Klebsiella variicola SHN-1. When the gene encoding pullulanase, containing the N-terminal signal sequence, was cloned into Escherichia coli BL21 (DE3), extracellular activity was detected up to 10 U/ml, a higher level compared with the results in published literature. Subsequently, the recombinant pullulanase was purified and characterized. The main end product from pullulan hydrolyzed by recombinant pullulanase was determined as maltotriose with HPLC, and hence, the recombinant pullulanase was identified as type I pullulanase, which could be efficiently employed in starch processing to produce maltotriose with higher purity and even to evaluate the purity of pullulan. To investigate the effect of signal peptide on secretion of the recombinant enzyme, the signal sequence was removed from the constructed vector. However, secretion of pullulanase in E. coli was not influenced, which was seldom reported previously. By localizing the distribution of pullulanase on subcellular fractions, the secretion of recombinant pullulanase in E. coli BL21 (DE3) was confirmed, even from the expression system of nonsecretory type without the assistance of signal peptide.