High-Molecular-Weight Plasmids Carrying Carbapenemase Genes blaNDM-1, blaKPC-2, and blaOXA-48 Coexisting in Clinical Klebsiella pneumoniae Strains of ST39.

BACKGROUND: Klebsiella pneumoniae, a member of the ESKAPE group of bacterial pathogens, has developed multi-antimicrobial resistance (AMR), including resistance to carbapenems, which has increased alarmingly due to the acquisition of carbapenemase genes located on specific plasmids. METHODS: Four clinical K. pneumoniae isolates were collected from four patients of a neuro-intensive care unit in Moscow, Russia, during the point prevalence survey. The AMR phenotype was estimated using the Vitec-2 instrument, and whole genome sequencing (WGS) was done using Illumina and Nanopore technologies. RESULTS: All strains were resistant to beta-lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and tetracyclines. WGS analysis revealed that all strains were closely related to K. pneumoniae ST39, capsular type K-23, with 99.99% chromosome identity. The novelty of the study is the description of the strains carrying simultaneously three large plasmids of the IncHI1B, IncC, and IncFIB groups carrying the carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2, respectively. The first of them, highly identical in all strains, was a hybrid plasmid that combined two regions of the resistance genes (blaOXA-48 and blaTEM-1 + blaCTX-M-15 + blaOXA-1 + catB + qnrS1 + int1) and a region of the virulence genes (iucABCD, iutA, terC, and rmpA2::IS110). CONCLUSION: The spread of K. pneumoniae strains carrying multiple plasmids conferring resistance even to last-resort antibiotics is of great clinical concern.

Genomic Analysis of a Hybrid Enteroaggregative Hemorrhagic Escherichia coli O181:H4 Strain Causing Colitis with Hemolytic-Uremic Syndrome.

Hybrid diarrheagenic E. coli strains combining genetic markers belonging to different pathotypes have emerged worldwide and have been reported as a public health concern. The most well-known hybrid strain of enteroaggregative hemorrhagic E. coli is E. coli O104:H4 strain, which was an agent of a serious outbreak of acute gastroenteritis and hemolytic uremic syndrome (HUS) in Germany in 2011. A case of intestinal infection with HUS in St. Petersburg (Russian Federation) occurred in July 2018. E. coli strain SCPM-O-B-9427 was obtained from the rectal swab of the patient with HUS. It was determined as O181:H4-, stx2-, and aggR-positive and belonged to the phylogenetic group B2. The complete genome assembly of the strain SCPM-O-B-9427 contained one chromosome and five plasmids, including the plasmid coding an aggregative adherence fimbriae I. MLST analysis showed that the strain SCPM-O-B-9427 belonged to ST678, and like E. coli O104:H4 strains, 2011C-3493 caused the German outbreak in 2011, and 2009EL-2050 was isolated in the Republic of Georgia in 2009. Comparison of three strains showed almost the same structure of their chromosomes: the plasmids pAA and the stx2a phages are very similar, but they have distinct sets of the plasmids and some unique regions in the chromosomes.

Rectal and Tracheal Carriage of Carbapenemase Genes and Class 1 and 2 Integrons in Patients in Neurosurgery Intensive Care Unit.

The spread of multidrug-resistant Gram-negative bacteria, which is associated with the distribution of beta-lactamase genes and class 1 and 2 integrons, is a global problem. In this study, in the Moscow neurosurgery intensive care unit (neuro-ICU), the high prevalence of the above-stated genes was found to be associated with intestinal and tracheal carriage. Seven-point prevalence surveys, which included 60 patients in the neuro-ICU, were conducted weekly in the period from Oct. to Nov. 2019. A total of 293 clinical samples were analyzed, including 146 rectal and 147 tracheal swabs; 344 Gram-negative bacteria isolates were collected. Beta-lactamase genes (n = 837) were detected in the isolates, including beta-lactamase blaTEM (n = 162), blaSHV (n = 145), cephalosporinase blaCTX-M (n = 228), carbapenemase blaNDM (n = 44), blaKPC (n = 25), blaOXA-48 (n = 126), blaOXA-51-like (n = 54), blaOXA-40-like (n = 43), blaOXA-23-like (n = 8), and blaVIM (n = 2), as well as class 1 (n = 189) and class 2 (n = 12) integrons. One extensively drug-resistant Klebsiella pneumoniae strain (sequence type ST39 and capsular type K23), simultaneously carried beta-lactamase genes, blaSHV-40 and blaTEM-1B, three carbapenemase genes, blaNDM, blaKPC, and blaOXA-48, the cephalosporinase gene blaCTX-M, and two class 1 integrons. Before this study, such heavily armed strains have not been reported, suggesting the ongoing evolution of antibiotic resistance.

Durable Superhydrophobic Coatings on Tungsten Surface by Nanosecond Laser Ablation and Fluorooxysilane Modification.

Tungsten is an attractive material for a variety of applications, from constructions in high-temperature vacuum furnaces to nontoxic shields for nuclear medicine, because of its distinctive properties, such as high thermal conductivity, high melting point, high hardness and high density. At the same time, the areas of the applicability of tungsten, to a large extent, are affected by the formation of surface oxides, which not only strongly reduce the mechanical properties, but are also prone to easily interacting with water. To alleviate this shortcoming, a series of superhydrophobic coatings for the tungsten surface was elaborated using the method of nanosecond laser treatment followed by chemical vapor deposition of hydrophobic fluorooxysilane molecules. It is shown that the durability of the fabricated coatings significantly depends on surface morphology and composition, which in turn can be effectively controlled by adjusting the parameters of the laser treatment. The coating prepared with optimized parameters had a contact angle of 172.1 ± 0.5° and roll-off angle of 1.5 ± 0.4°, and preserved their high superhydrophobic properties after being subjected to oscillated sand abrasion for 10 h, continuous contact with water droplets for more than 50 h, and to several cycles of the falling sand test.

Multidrug-Resistant Klebsiella pneumoniae Causing Severe Infections in the Neuro-ICU.

The purpose of this study was the identification of genetic lineages and antimicrobial resistance (AMR) and virulence genes in Klebsiella pneumoniae isolates associated with severe infections in the neuro-ICU. Susceptibility to antimicrobials was determined using the Vitek-2 instrument. AMR and virulence genes, sequence types (STs), and capsular types were identified by PCR. Whole-genome sequencing was conducted on the Illumina MiSeq platform. It was shown that K. pneumoniae isolates of ST14K2, ST23K57, ST39K23, ST76K23, ST86K2, ST218K57, ST219KL125/114, ST268K20, and ST2674K47 caused severe systemic infections, including ST14K2, ST39K23, and ST268K20 that were associated with fatal incomes. Moreover, eight isolates of ST395K2 and ST307KL102/149/155 were associated with manifestations of vasculitis and microcirculation disorders. Another 12 K. pneumoniae isolates of ST395K2,KL39, ST307KL102/149/155, and ST147K14/64 were collected from patients without severe systemic infections. Major isolates (n = 38) were XDR and MDR. Beta-lactamase genes were identified: blaSHV (n = 41), blaCTX-M (n = 28), blaTEM (n = 21), blaOXA-48 (n = 21), blaNDM (n = 1), and blaKPC (n = 1). The prevalent virulence genes were wabG (n = 41), fimH (n = 41), allS (n = 41), and uge (n = 34), and rarer, detected only in the genomes of the isolates causing severe systemic infections-rmpA (n = 8), kfu (n = 6), iroN (n = 5), and iroD (n = 5) indicating high potential of the isolates for hypervirulence.

Carriage of Capsular Serotype K1 Klebsiella pneumoniae Sequence Type 23 Strains in Healthy Microbiology Laboratory Staff in Russia.

Klebsiella pneumoniae causes both nosocomial and community-associated infections. Among the hypervirulent K. pneumoniae (hvKP) isolates, K1 is the most common capsular serotype. Here, we report the draft genome sequences of 3 K1-type (sequence type 23) K. pneumoniae strains isolated from healthy microbiology laboratory staff in Russia.

Bioimpedance Spectroscopy: Basics and Applications.

In this review, we aim to introduce the reader to the technique of electrical impedance spectroscopy (EIS) with a focus on its biological, biomaterials, and medical applications. We explain the theoretical and experimental aspects of the EIS with the details essential for biological studies, i.e., interaction of metal electrodes with biological matter and liquids, strategies of measurement rate increasing, noise reduction in bio-EIS experiments, etc. We also give various examples of successful bio-EIS practical implementations in science and technology, from whole-body health monitoring and sensors for vision prosthetic care to single living cell examination platforms, virus disease research, biomolecules detection, and implementation of novel biomaterials. The present review can be used as a bio-EIS tutorial for students as well as a handbook for scientists and engineers because of the extensive references covering the contemporary research papers in the field.

FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho.

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

Structures of ligand-occupied ß-Klotho complexes reveal a molecular mechanism underlying endocrine FGF specificity and activity.

The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or ß-Klotho receptors. Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to ß-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a S-P-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing ß-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligand-occupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses.

Charge-mediated proteasome targeting.

A majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin (Ub), representing a universal degradation signal operated by the ubiquitination system. Ub-independent proteasome targeting is rationalized by the existence of 2 types of direct proteasome signals (DPSs), specific amino acid sequences or post-translational modifications, which are recognized by proteasome regulatory subunits. Historically, the first type was shown to exist in ornithine decarboxylase, whereas acetylation of core histones recently was reported as a second type of DPS. Here we declare a third type, representing charge-mediated DPS. This discovered DPS may be classified as a monopartite composition- but not sequence-dependent element of ∼70 Å in length enriched in basic and flexible amino acids. This type of degradation signal, which may be provided by cationic chemicals, is most efficiently engaged by proteasomes capped with regulator (REG)α or REGγ in an ATP-independent manner. Taken together, our findings suggest a novel modality of proteasome-substrate interrelation bypassing ubiquitination.-Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V., Belogurov, A. Charge-mediated proteasome targeting.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells.

The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2'-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases.

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and ß1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, ß1i is increased in resident CNS cells, whereas ß5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived ß1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the ß1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

Glatiramer acetate and nanny proteins restrict access of the multiple sclerosis autoantigen myelin basic protein to the 26S proteasome.

We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.