HELICOBACTER PYLORI BIOFILM IN VITRO AND IN VIVO Zhukhovitsky V. G.1, 2, 3, Smirnova T. A.1, Shevlyagina N. V. 1, Korzheva I. Yu.2, Didenko L. V. 1, Gintsburg A. L.1, 3 1 Gamaleya Federal Research Centre for Microbiology and Immunology of the Ministry of Public Health 2 Botkin Hospital of Moscow Department of Public Health 3 Sechenov the First Moscow Medical University of the Ministry of Public Health

An issue on the cellular forms that ensure survival of pseudomonads is important due to wide occurrence of these bacteria in the environment and their role for clinical microbiology. The present work demonstrates the high survival potential of Pseudomonas aurantiaca and P. аeruginosa in the mass of exopolymers produced by cells. Exopolymer formation occurred only during incubation of the post-stationary phase cultures of P. aurantiaca (at 4°C) and P. aeruginosa (at 4 and 20°C). After storage for 1.5–12 months, the number of colony-forming units in the exopolymer was 30 to 68% of the viable cell titer in stationary-phase cultures. Antibiotic-tolerant persister cells that were revealed in the exopolymer cultures after treatment with ciprofloxacin (2.5–100 μg/mL) were more resistant to the antibiotic than persisters in suspension cultures, with the threshold doses of 25 and 2.5 μg/mL, respectively. The cells embedded in the exopolymer were found to be more resistant to 5-min heating at 60–70°C than the vegetative cells of suspension cultures, which did not survive such heat treatment conditions. Electron microscopic investigation revealed morphological heterogeneity of exopolymer-embedded pseudomonads, including the presence of the cells similar to cystlike dormant forms. The populations developing on solid media inoculated with the exopolymer mass with cells were found to contain 1.5 to 2 orders of magnitude more persisters tolerant to high ciprofloxacin doses (25 μg/mL for P. aurantiaca and 100 μg/mL for P. aeruginosa) than the populations developing after inoculation with second-transfer vegetative cells of the cells of planktonic cultures. The results obtained improve our understanding of pseudomonad survival in the environment.

Using scanning electron microscopy, the ability of reference and freshly isolated Helicobacter pylori strains to form biofilm under cultivation on abiotic surfaces and in natural conditions was studied. It was shown that both types of strains were capable of biofilm formation in vitro, although reference strains synthesized less pronounced exocellular matrix and flagella than freshly isolated strains. Bio-film also was detected in specimens of antral mucosa under duodenal ulcer. Most often Helicobacter pylori biofilms were detected near the mouths of the gastric glands. The structure of matrix surface of such biofilms looked inhomogeneous in different parts of the epithelial layer.

V.M. Elinson, L.V. Didenko, N.V. Shevlyagina, G.A. Avtandilov, A.N. Lyamin, O.A. Silnitskaya Moscow Aviation Institute (National Research University), 125993, Volokolamskoe Shosse, 4, Moscow, Russia Gamaleya Research Center for Epidemiology and Microbiology Ministry of Healthcare, 123098, Gamaleya str,18, Moscow, Russia Moscow State Medical and Stomatological University Ministry of Healthcare, Delegatskaya str, 20 bld.1,Moscow, Russia

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.

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.

Research on staphylococci research is important because of their ability to cause such severe infections as soft tissue 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 weakened immune system. The mechanisms of the CNS pathogenicity are understood insufficiently. The goal of this work was to evaluate the potential pathogenicity of clinical CNS strains based on their capacity to form biofilms and their character of interaction with the human cells through an example of HT-29 cell culture. The research was carried out on the laboratory strain S. aureus ATCC 29213 and clinical strains S. haemolyticus SH39 and S. epidermidis SE 36-1, which were isolated from neonatal autopsy materials. The visual tests of biofilm formation by each strain and testing the impact of the strains on HT-29 cell culture were carried out in this work. Two CNS species form biofilms with a higher rate than S. aureus. Upon incubation for 2 h of HT-29 cells with the staphylococcus strains tested in this work, adhesion of bacteria on the cells’ surfaces was observed. The adhesion was most pronounced in the case of S. aureus ATCC 29213 and S. haemolyticus SH39. Upon 3 h of incubation with S. aureus ATCC 29213 and S. haemolyticus SH39, the destruction of a monolayer of HT-29 cells was observed. The incubation for 24 h with three strains tested in this work caused the complete destruction of the monolayer of HT-29 cells. The maximal toxic effect on HT-29 cells was inherent in S. haemolyticus SH39 strain. The cumulative results obtained in this work indicate the presence of the pathogenicity factors in S. haemolyticus SH39 strain, and their molecular nature needs to be researched further.

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.

Spore accumulation in Brevibacillus laterosporus in a liquid nutrient medium under optimal conditions during stationary cultivation was investigated. The structure of floating biofilms was studied by transmission and scanning electron microscopy. It was shown that B. laterosprus cells formed a biofilm within 96 h of incubation. The accumulation of spores and individual vegetative cells in the biofilm was confirmed by light microscopy. The spore ultrastructure was shown to depend on the richness of the media. As was shown for the first time, B. laterosprus had an unusual morphology when grown in a yeast polysaccharide medium. A difference between B. laterosprus and Bacillus subtilis biofilms concerning the vegetative cells and spores was observed. In B. subtilis biofilm, the vegetative cells are numerous and devoid of the defects that are characteristic of B. laterosprus. The results may be helpful in optimizing bacilli-based biotechnological processes aimed at improving the yield of target products.

The authors examined 40 muscle biopsy specimens taken from patients with neuromuscular symptoms when the diagnosis was unestablished or presumptive. Eighteen of them exhibited foci of muscle fiber damage with the presence of spirochete-like structures in the semithin tissue sections. Electron microscopy of these areas detected Borrelia as vegetative and diverse L-forms. Immunocytochemical techniques using antibodies to Borrelia burgdorferi antigens confirmed that the spirochetes belonged to this species. This allows one to consider borreliosis as an etiological or complicating factor of neuromuscular pathology and to recommend the above morphological methods for the diagnosis of neuromuscular diseases of unknown origin.

Clinical material obtained surgically from patients with kidney-stone disease (KSD) was tested for the composition and state of stone microflora using the standard microbiological methods, PCR, and modern microscopy technologies. It was demonstrated that about 50% of stones in patients with KSD were infected with various infection agents, as observed using the standard microbiological and molecular-genetic meth-ods. The percentage of Mycoplasma hominis detected using the cultural method is lower than that provided by PCR, which is due to the difficulty of isolation and cultivation, as well as DNA fragments of mycoplasma observed after antibiotic therapy. Studies based on the modern microscopy technologies revealed that micro-organisms on the kidney stone surface formed multispecies biofilms.

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.

Comparative analysis of the structure of the surface of different materials showed that the most resistant to biodegradation caused by staphylococci, is Ftoraks and silicone. The material’s resistance to biodegradation is determined by the chemical composition of the material, Ftoraks contains fluorine, the element which has an antibacterial effect. As shown previously, the distribution of fluorine in this material is uneven. In those parts of the surface, where is recorded the presence of fluoride, there is no adhesion ofmicroorganisms and therefore its destruction (10). Silicone is composed of the organosilicon compounds, and, apparently, those organic components included in it, are not destroyed by enzymes, acids or alkalis produced by bacteria.

Study the processes of bacterial colonization and persistence on the surface of polymeric plastics. Plates (1x1 cm) of polymer plastics (polyurethane and acrylic) were used («Dentalur» Russia, «Ftoraks» «Plastic colorless» Stoma, Ukraine). These materials are applied for manufacturing medical dentures. Polymeric plates were incubated in the Luria-Bertani nutrient broth with clinical isolates of P. aeruginosa, S. aureus during 24, 48 hours, 7 and 14 days, 1, 5 and 3 months at a temperature of 37˚C. After incubation, the samples were fi xed in 10% neutral formalin solution. Interaction of bacteria with polymeric materials was studied using scanning electron microscopy (SEM). Traditional SEM dehydration of samples by alcohols or acetone was not carried out, that allowed to study the native structure of extracellular matrix. The stages of interaction between bacteria and surfaces of polymers were investigated. Bacterial biofi lms on the polymeric surfaces provoked their biodestruction changes. The possibility of long-term persistence of human pathogenic microorganisms on artifi cial prostheses is discussing.

This research was conducted on renal concretions from patients suffering from nephrolithiasis, a pathology caused by the presence of kidney stones. This disease provokes inflammation of the interested area, pain and functional alterations of the organ. Microorganisms play a leading role in stones formation being at the base of septic complications. Urease producing microorganisms (Escherichia coli among them) are typical microorganisms participating in stones formation. Scanning Electron Microscope (SEM) and Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) techniques show that microorganisms remain in renal calculi for a long time and produce biofilm, a mucoid matrix with several physiochemical microenvironments in which bacteria act as a community. Bacteria, organized in microcolonies within the biofilm, cause infectious processes and provoke commissures formations, which fix stones to kidneys. Electron microscopy observation of stones from patients suffering from nephrolithiasis who underwent surgery or lithotripsy shows the presence of collagen fibers on the concrements. This suggests that it is required a proper surgical methodology for calculi removal, in order to prevent the infection from spreading and to avoid a relapse of nephrolithiasis.