[Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy].

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

A novel antivirulent compound fluorothiazinone inhibits Klebsiella pneumoniae biofilm in vitro and suppresses model pneumonia.

The problematic treatment of infections caused by multiple-resistant Klebsiella, especially in ICU, is the leading cause of prolonged hospitalization and high mortality rates. The use of antibiotics for the prevention of infections is considered unreasonable as it may contribute to the selection of resistant bacteria. In this regard, the development of drugs that will be effective in preventing infection during various invasive procedures is extremely necessary. We have shown that the developed innovative antibacterial compound fluorothiazinone (FT) that suppresses the formation of biofilms is effective in the prevention of a model pneumonia caused by a multi-resistant clinical K. pneumoniae isolate. Prophylactic use followed by treatment with FT in mice with acute pneumonia modulates the local innate immune response without suppressing protective properties in the early stages of infection, while contributing to a decrease in the bacterial load in the organs and preventing lethal pathological changes in the lungs at later stages of K. pneumoniae infection. Further development of such antivirulence drugs and their use will reduce morbidity and mortality in nosocomial infections, as well as reduce the number of antibiotics used.

[Antibacterial effect of the antiseptic picloxydine dihydrochloride on conjunctival isolates of gram-negative bacteria]. / Antibakterial'noe deistvie antiseptika pikloksidina digidrokhlorida na kon"yunktival'nye izolyaty gramotritsatel'nykh bakterii.

The preoperative and postoperative use of antiseptics can be an alternative to antibiotics in repeated courses of anti-VEGF therapy for reducing the risk of developing antibiotic resistance in eye microflora. Among gram-negative bacteria, the most frequently isolated pathogen that causes eye infections is Pseudomonas aeruginosa, which is characterized by reduced sensitivity to antibiotics and disinfectants. PURPOSE: To study the effect of the antiseptic picloxydine dihydrochloride on the gram-negative bacteria Escherichia coli, Pseudomonas luteola and P. aeruginosa isolated from the conjunctiva. MATERIAL AND METHODS: The identification of bacterial isolates and study of their sensitivity to antibiotics were carried out using the automated bacteriological analyzer BD Phoenix 100. To determine the bactericidal concentration, the method of serial dilutions of the antiseptic in a liquid nutrient medium was used. The binding of cationic molecules of picloxydine dihydrochloride to bacterial cells was detected by neutralizing the bacterial surface with increasing amounts of antiseptic, and measuring the zeta potential on the Zetasizer Nano ZS analyzer. The ultrastructure of bacterial cells was studied using the two-beam scanning ion-electron microscope Quanta 200 3D. RESULTS: The most resistant was P. aeruginosa. The interaction mechanism of picloxydine dihydrochloride with bacterial cells includes electrostatic binding of positively charged antiseptic molecules to negatively charged cell walls. Picloxydine dihydrochloride has a destructive effect on the bacterial cell wall and plasma membrane, which leads to cell lysis and release of intracellular components. CONCLUSION: Picloxydine dihydrochloride exhibits bactericidal activity against gram-negative conjunctival isolates and is promising for preventive use during repeated courses of intravitreal injections.

Morphology and Ultrastructure of Group A Streptococcus Biofilms.

Light and electron microscopy enables researchers to study the ultrastructure of GAS biofilms formed on abiotic surfaces. Chains of streptococci surrounded by a bluish film are seen under a light microscope after alcian blue staining of preparations grown on coverslips. The extracellular matrix (indicator of biofilm maturity) becomes visible on ultrathin sections in transmission electron microscopy after additional staining with alcian blue; filamentous structures, characteristic of biofilm, are observed in intercellular spaces. The data obtained by scanning electron microscopy also demonstrate the presence of biofilm.

Optical and Electron Microscopic Study of the Morphology and Ultrastructure of Biofilms Formed by Streptococcus pyogenes.

Our study confirmed the capacity of S. pyogenes strains to form biofilms on abiotic surfaces. Chains of streptococci surrounded by bluish film were seen under a microscope after alcian blue staining of the preparations grown on slides. On ultrathin sections in transmission electron microscope, the extracellular matrix (indicator of biofilm maturity) became visible after staining with alcian blue. Microscopy of the sections shows structures characteristic of a biofilm in spaces between the cells. Scanning electron microscopy also demonstrates the presence of a biomembrane. Importantly that type 1M strain forming in fact no membranes when cultured on plastic plates (Costar) formed biofilms on the glass. It seems that the conditions for the biofilm formation on the plastic and on the glass differ, due to which the exopolymeric matrices formed on different surfaces vary by biochemical composition.

MORPHOLOGICAL ANALYSIS OF HEPATITIS B VIRUS WITH ESCAPE MUTATIONS IN S-gene G145R AND S143L.

BACKGROUND: In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. OBJECTIVES: Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. METHODS: Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. RESULTS: The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. CONCLUSION: Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

Colonization by Staphylococcus aureus of Nano-Structured Fluorinated Surfaces, Formed by Different Methods of Ion-Plasma Technology.

Colonization of fluorinated surfaces produced by ion-plasma technology by Staphylococcus aureus was studied by scanning electron microscopy and surface energy analysis. It was shown that the intensity of colonization was determined by the surface relief and fluorine content. Formation of nanostructured surfaces accompanied by a sharp decrease in the surface energy prevented adhesion of Staphylococcus aureus cells to the fluorine-containing surface.

[ABILITY OF STAPHYLOCOCCUS OF VARIOUS STRAINS TO CREATE BIOFILMS AND THEIR EFFECT ON HUMAN BODY CELLS].

The urgency of the staphylococcus research is due to its ability to cause severe infections: softtissue infections, endocarditis, sepsis, toxic shock syndrome, and food poisoning. Coagulase-positive Staphylococcus aureus is the main infection agent of intrahospital infections. This agent has many factors of pathogenicity, which are well known. Among the coagulase-negative staphylococcus (CNS) strains, S. haemolyticus and S. epidermidis are clinically important, because they cause infections in patients with weak immune system. The mechanisms of the CNS pathogenicity are insufficiently understood. The goal of this work was to evaluate the potential pathogenicity of clinical strains of CNS from their capacity to create biofilms and the character of their interaction with human body cells by the example of the HT-29 cell culture. The research was carried out in laboratory strain S. aureus ATCC 29213 and clinical strains S. haemolyticus SH39, S. epidermidis SE36-1 isolated from the neonatal autopsy materials. The visual tests of biofilm formation by each strain and testing of the impact of the strains on the cell culture HT-29 was carried out in this work. The two species of CNS form biofilms at a higher rate than S. aureus. Upon incubation for 2 h of HT-29 cells with staphylococcus strains tested in this work, adhesion of bacteria on cell surface was observed. The adhesion was most pronounced in case of S. aureus ATCC 29213 and S. haemolyticus SH39. Upon 3 h of incubation with S. aureus ATCC 29213 and S. haemolyticus SH39, destruction of cell HT-29 monolayer was observed. The incubation for 24 h with the 3 strains tested in this work caused complete destruction of cell HT-29 monolayer. The maximal toxic effect on HT-29 cells was inherent in the strain S. haemolyticus SH39. The aggregate of the results obtained in this work indicates the presence of the pathogenicity factors in the strains S. haemolyticus SH39, which require additional research.

[A porous coral-like structure - a new insight into the morphology of the inner limiting membrane of the retina?] / Poristaya korallovidnaya struktura ­ novoe predstavlenie o morfologii vnutrennei pogranichnoi membrany setchatki?

To date, the internal limiting membrane (ILM), specifically, the side facing the retina, has never been studied by two parallel, mutually complementary methods. This is an attempt to explain favorable results of ILM peeling in various macular pathologies. AIM: By employing scanning (SEM) and transmission electron microscopy (TEM), to identify morphological features of epiretinal samples removed during vitrectomy in patients with lamellar macular hole (LMH) or epiretinal membrane (ERM). MATERIAL AND METHODS: We studied 23 eyes of 23 patients divided into two groups. The first group (13 samples, 11 eyes) consisted of patients with LMH; the second (12 samples, 12 eyes) - with ERM. The surgeries yielded a total of 21 epiretinal samples peeled simultaneously with the ILM and 4 epiretinal samples (2 eyes) peeled in two parts, the second part containing the ILM. One half of the samples was studied by SEM without prior dehydratation, the other - by TEM. RESULTS: The study revealed a high degree of ultrastructural similarity between the two groups of ILM samples. Judging from SEM findings, two sides of the membrane were clearly identified. Porous coral-like structures (PCS) were discovered on the side facing the retina. TEM in the area of PCS discovered parallel arrangement of multiple Muller cell (MC) bodies and processes separated by wide layers of the intercellular matrix. The vitreal side of all ILM samples was notable for numerous fibroblast-like cells. Many variously shaped petrified structures were found on both sides of the membrane. CONCLUSION: During the so called ILM peeling, the surgeon removes a layered structure that includes the basal membrane of MC, cells and fibers attached to its vitreal side, and one more layer comprised by PCS and rather readily torn off from the main massif. The functional significance of this previously unknown structure as well as the effect of its partial removal during surgical manipulations with neurosensory retina in the macular region is yet to be investigated.

[Efficacy of polyprenyl phosphates in the experimental genital herpes model].

An experimental model of the primary genital herpes (herpes simplex type 2, HSV-2) in the female guinea pigs was suggested to study the infectious process activity of polyprenyl phosphates (PPP) and PPP+acyclovir (AC) complex treatment. The morphofunctional features of the guinea pig ovaries were studied in the control and experimental groups (the latter were inoculated with PPP and/or AC as a primary infection treatment) at the stage of the recurrent genital herpes aggravation. It was shown that in the case of combined PPP +AC use significant changes in the disease symptoms were observed, as well as a decrease in the infectious process activity and duration, and positive remote effect on the ovarian morphophysiology.

[STUDY OF COLONIZATION PROCESSES AND PERSISTENCE OF MICROORGANISMS IN ARTIFICIAL MATERIALS FOR MEDICAL USE].

AIM: Study processes of microbial colonization and persistence of microorganisms in polymer materials for medical use. MATERIALS AND METHODS: Samples (1 x 1 cm plates) of polymer plastics for production of removable dental prosthesis based on polyurethane and acryl were used, that were incubated with clinical isolates of Pseudomonas aeuruginosa, Staphylococcus aureus in Luria-Bertani broth nutrient media for 24, 48 hours and 7, 14 days and for 1, 5 and 3 months at a temperature of 37 degrees C. Dynamics of interaction process of microorganisms with polymer materials were studied using scanning electron microscope Quanta 200 3D (FEI Company, USA). The samples were fixated after incubation with 10% of neutral formaldehyde, dehydration with alcohols or acetone, typical for SEM, was not carried out, that allowed to conserve the native structure of the samples, including exo-cell matrix of biofilms. RESULTS: Electron-microscopical data on stages of interaction of bacteria with the surface of medical plastics were obtained. Biofilms were shown to be formed on abiotic surfaces and biodestructive changes of plastics appeared. A question on the possibility of prolonged persistence of pathogenic for human microorganisms in artificial prosthesis is discussed. CONCLUSION: The developed experimental model of formation of biofilm on abiotic surfaces could be the basis for carrying out studies directed on the fight with biofilms, by using SEM.

Morphofunctional changes in guinea pig ovaries in experimental chronic herpesvirus infection.

The ovaries of adult guinea pigs infected with herpesvirus-2 were examined by light microscopy under conditions of latent infection and relapse of infectious process. Morphofunctional changes in the ovarian follicles at all stages of folliculogenesis and in ovarian stroma were caused by the negative effects of type 2 herpes simplex virus.