Abstract OR-5: Helicobacter pylori Surface Structures

Background: Helicobacter pylori, which infects at least half of the human population, is an etiopathogenetic factor in the development of chronic gastritis (CG), gastric and duodenal ulcer disease (UD), and is also considered a risk factor in the occurrence of some forms of stomach cancer. Various surface structures of H. pylori are important pathogenic factors. Methods: Reference (NCTC 11637, NCTC 11639) and freshly isolated H. pylori strains as well as samples of the gastric mucosa were examined using transmission (TEM) and scanning electron microscopy (SEM) by means of “JEM-100 В” (JEOL, Japan) and “Quanta 200 3D” (FEI Company, USA) systems, respectively. Samples for SEM fixed with formalin and not subjected to dehydration were sprayed with an electrically conductive layer of gold. Samples for TEM were prepared using negative contrast (NC) with ammonium molybdate and ultrathin sections (US) contrasted by Reynolds; fixation by Ito-Karnovsky was used in both cases. Accelerating voltage at TEM and SEM was 80 and 10 kV, respectively. Results: Three types of H. pylori surface structures were found: flagella, fimbria, and vesicles. Flagella were found both in the material of cultures maintained in vitro and in each native sample of the gastric mucosa. SEM images suggest the involvement of flagella in biofilm formation. On longitudinal sections of the flagellum, the outer electron-dense layer and the inner content in the form of a filament were visualized; on cross sections, the flagellum looked like an annular structure with a centrally located point accumulation of electron-dense matter. Paradoxically, the H. pylori forms with a defective cell wall also possessed flagella. Freely located flagellar sheaths without central filament were often found in vitro only. Fimbriae were found exclusively in negatively contrasted pure bacterial cultures material. In addition, vesicles detaching from the surface of bacterial cells were found both in the material of aging H. pylori pure cultures and in native samples of the gastric mucosa under CG and UD. A small part of the vesicles retains their connection with the cell wall, while detached vesicles are present in excess in the surrounding space. Massive detachment of vesicles leads to the formation of spheroplasts, devoid of an outer membrane. Conclusion: H. pylori has a representative set of surface structures that play an important role in the onset and development of the infectious process in the gastroduodenal area

Sensitivity of antibiotic resistant coagulase-negative staphylococci to antiseptic piсloxydin

Background. Coagulase-negative staphylococci (CNS), primarily Staphylococcus epidermidis, predominate in the normal microflora of the eye. However, due to irrational antibiotic therapy, resistant strains are widely distributed among CNS. Aim. To study the sensitivity of the antibiotic resistant CNS isolates to picloxydine, an antiseptic. Methods. The species, sensitivity to antibiotics and picloxydine were determined for 39 isolates of bacteria obtained from the conjunctival swabs. The cells morphology under the antiseptics influence was studied by electron microscopy. Results. 33 isolates of S. epidermidis (17 sensitive or resistant to drugs of no more than 2 classes of antibiotics and 16 MDR), 2 S. haemolyticus (1 resistant to 2 classes of antibiotics and 1 MDR), 3 S. hominis (1 sensitive and 2 MDR), 1 S. caprae (MDR) were characterized. In in vitro tests, picloxydine showed high efficiency in suppressing the growth of staphylococci regardless of their sensitivity to antibiotics, as well as bactericidal activity at concentrations of 15.631.2 g/ml, close to those of chlorhexidine. At these concentrations, the antiseptic had a destructive effect on the surface structures of bacterial cells. Conclusion. The picloxydine antiseptic is equally effective against antibiotic- sensitive and antibiotic-resistant coagulase-negative staphylococci

CHANGE IN CONTENT OF GLYCOPROTEINS ON THE SURFACE OF ENDOTHELIAL CELL CULTURE EA.HY 926 AND INTIMA OF INTERNAL CAROTID ARTERIES UNDER THE INFLUENCE OF MAGNESIUM OROTATE

Aim. To study changes in the glycoproteins content in the glycocalyx of endothelial cells under the influence of magnesium orotate.Material and methods. Endotheliocytes of cell culture EA.hy 926 were examined before  and after addition of the magnesium orotate to the culture medium. Intimal endothelium of internal carotid arteries, fragments of which were obtained by sequential execution of bilateral resection of the arteries before  and after course of treatment with magnesium orotate was also investigated. Anthony’ method (1931), designed for the study of glycoproteins in the bacteria capsule and adapted by L.V. Didenko (2013) for eukaryotes was used to detect glycoproteins. The scanning electron microscopy with simultaneous x-ray microanalysis was applied.Results. Total number of Cu2+-signals per one scanning field in the mapping of samples for Cu2+ as the indirect index of proteoglycan content in EA.hy 926 endothelial cell culture before incubation with magnesium orotate was 6928±124, and after incubation with magnesium orotate – 7592±131. Number of Cu2+-signals on the surface of the internal carotid artery intima before treatment with magnesium orotate was 5015±407, and after treatment – 6100±152 per one scanning field.Conclusion. A significant increase in the content of glycoproteins on the surface of EA.hy926 endothelial cell culture (+10%) and internal carotid artery intima (+22%) under the influence of magnesium orotate was found

Смешанные бактериально-вирусные инфекции легких у пациентов с муковисцидозом

The main cause of death in patients with cystic fibrosis (CF) is infectious process in the lungs, in particular, chronic lung infections caused by various pathogens, most often a combination of bacteria, fungi, or viruses. Data on mixed bacterial and viral-bacterial infections from domestic and foreign sources are fragmentary and sparse. The dominant associations of bacterial and viral pathogens in patients with cystic fibrosis have not been studied properly, and data on their epidemiological significance are lacking. The aim of this study was to assess the prevalence of bacterial and viral infections in patients with cystic fibrosis and to substantiate the need for the development of virological monitoring. Methods. Biomaterials from the respiratory tract of CF patients (409 children and 160 adults with CF) examined from 2006 to 2022 were used. The study was carried out using bacteriological methods, molecular genetic methods (RT-PCR) and MALDI-TOF mass-spectrometry. Results. Microflora of the respiratory tract was shown to be mixed in 2/3 patients with CF. The microflora of the lungs of children with CF is a dynamic community of microorganisms with high diversity and variability. In adult patients, associations of microorganisms are more common than in children, but the composition of associations is less diverse. We isolated about 40 species of bacteria from adult patients and more than 85 species from children in our sample. NFMO prevailed, including Burkholderia cepacia complex, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Achromobacter ruhlandii, Stenotrophomonas maltophilia, and Staphylococcus aureus, Candida albicans, Aspergillus spp. Real-time PCR showed the presence of rhinovirus RNA in 10% of samples obtained from children and 12.9% from adults with cystic fibrosis. Conclusion. Our results indicate the need for continuous monitoring of the lung microflora in patients with CF, including testing for viruses.Основной причиной смерти больных муковисцидозом (МВ) являются инфекционные процессы в легких, в частности, хронические инфекции легких (ХИЛ), вызываемые различными возбудителями, среди которых выявлены доминирующие, чаще всего в виде смешанного инфицирования, – бактериями, грибами, вирусами. Данные о смешанных бактериальных и бактериально-вирусных инфекциях (БВИ), полученных из отечественных и зарубежных источников, фрагментарны и немногочисленны. В настоящее время доминирующие ассоциации возбудителей бактериальной и вирусной природы у пациентов с МВ изучены недостаточно, отсутствуют данные об их эпидемиологической значимости. Целью работы являлась оценка распространенности БВИ и обоснование необходимости разработки системы вирусологического мониторинга у пациентов с МВ. Материалы и методы. Материалом для исследования послужили биоматериалы, полученные из дыхательных путей детей (n = 409) и взрослых (n = 160) больных МВ, обследованных в 2006–2022 гг. Исследование проводилось с помощью бактериологических, молекулярно-генетических методов (полимеразная цепная реакция (ПЦР) в режиме реального времени (РВ) с обратной транскрипцией; времяпролетная масс-спектрометрия с матрично-ассоциированной лазерной десорбцией / ионизацией (Matrix-Assisted Laser Desorption / Ionization (MALDI) Time-Of-Flight (TOF)). Результаты. Показано, что микрофлора дыхательных путей у пациентов с МВ в 2/3 случаев является смешанной. Микрофлора легких у детей с МВ представляет собой динамичное сообщество микроорганизмов, характеризуемых большим разнообразием и изменчивостью. У взрослых больных ассоциации микроорганизмов встречаются чаще, чем у детей, но менее разнообразен состав ассоциаций. От выборки взрослых больных выделены около 40 видов бактерий (от детей – > 85 видов), среди который преобладали неферментирующие грамотрицательные микроорганизмы: Burkholderia cepacia complex, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Achromobacter ruhlandii, Stenotrophomonas maltophilia, а также Staphylococcus aureus, Candida albicans и Aspergillus spp. По результатам ПЦР РВ показано наличие РНК риновируса в 10 % образцов, полученных от детей, и 12,9 % – от взрослых пациентов с МВ. Заключение. По результатам исследования обоснована необходимость постоянного мониторинга микрофлоры легких у больных МВ, включая исследование на вирусы

Targeting of Silver Cations, Silver-Cystine Complexes, Ag Nanoclusters, and Nanoparticles towards SARS-CoV-2 RNA and Recombinant Virion Proteins

Background: Nanosilver possesses antiviral, antibacterial, anti-inflammatory, anti-angiogenesis, antiplatelet, and anticancer properties. The development of disinfectants, inactivated vaccines, and combined etiotropic and immunomodulation therapy against respiratory viral infections, including COVID-19, remains urgent. Aim: Our goal was to determine the SARS-CoV-2 molecular targets (genomic RNA and the structural virion proteins S and N) for silver-containing nanomaterials. Methods: SARS-CoV-2 gene cloning, purification of S2 and N recombinant proteins, viral RNA isolation from patients’ blood samples, reverse transcription with quantitative real-time PCR ((RT)2-PCR), ELISA, and multiplex immunofluorescent analysis with magnetic beads (xMAP) for detection of 17 inflammation markers. Results: Fluorescent Ag nanoclusters (NCs) less than 2 nm with a few recovered silver atoms, citrate coated Ag nanoparticles (NPs) with diameters of 20–120 nm, and nanoconjugates of 50–150 nm consisting of Ag NPs with different protein envelopes were constructed from AgNO3 and analyzed by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible light absorption, and fluorescent spectroscopy. SARS-CoV-2 RNA isolated from COVID-19 patients’ blood samples was completely cleaved with the artificial RNase complex compound Li+[Ag+2Cys2−(OH−)2(NH3)2] (Ag-2S), whereas other Ag-containing materials provided partial RNA degradation only. Treatment of the SARS-CoV-2 S2 and N recombinant antigens with AgNO3 and Ag NPs inhibited their binding with specific polyclonal antibodies, as shown by ELISA. Fluorescent Ag NCs with albumin or immunoglobulins, Ag-2S complex, and nanoconjugates of Ag NPs with protein shells had no effect on the interaction between coronavirus recombinant antigens and antibodies. Reduced production of a majority of the 17 inflammation biomarkers after treatment of three human cell lines with nanosilver was demonstrated by xMAP. Conclusion: The antiviral properties of the silver nanomaterials against SARS-CoV-2 coronavirus differed. The small-molecular-weight artificial RNase Ag-2S provided exhaustive RNA destruction but could not bind with the SARS-CoV-2 recombinant antigens. On the contrary, Ag+ ions and Ag NPs interacted with the SARS-CoV-2 recombinant antigens N and S but were less efficient at performing viral RNA cleavage. One should note that SARS-CoV-2 RNA was more stable than MS2 phage RNA. The isolated RNA of both the MS2 phage and SARS-CoV-2 were more degradable than the MS2 phage and coronavirus particles in patients’ blood, due to the protection with structural proteins. To reduce the risk of the virus resistance, a combined treatment with Ag-2S and Ag NPs could be used. To prevent cytokine storm during the early stages of respiratory infections with RNA-containing viruses, nanoconjugates of Ag NPs with surface proteins could be recommended

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows