Features of formation of Yersinia enterocolitica biofilms

Aim: The work aimed to study the morphology of colonies and their comparison by features of the formation of Yersinia enterocolitica biofilms. Materials and Methods: Bacteria were cultured on a Yersinia Selective Agar medium ("CIN-agar") at 28°C for 24 h. The microorganisms were grown in meat-peptone broth with 1.0% glucose to measure the absolute values of the optical density of the culture. The optical density of the liquid was determined in a microplate photometric analyzer Immunochem-2100 (HTI, USA) at a wavelength of 490 nm. For the study of biofilms, the specimens were fixed for 3-5 h in pairs of 25.0% solution of glutaraldehyde (according to DV), and pairs of a 1.0% aqueous solution of osmic acid (OSO4) were used for contrasting for 2-3 min. The specimens were examined with stereoscopic microscopy "BIOMED MS-1 Stereo" (Russia) and scanning electron microscope "TM 3030 plus" (Holland). Results: With stereoscopic microscopy of the colonies of Y. enterocolitica, the S-forms had an elevated intensely colored center, radial striation along the periphery, smooth edges, d ≤ 1.0 mm. R-form colonies had a dark color and a dry surface, were tuberous and had a dense center with a peripheral ridge, rugged edges, d ≥ 1.5 mm. The optical density of the Y. enterocolitica S-form showed that this type of microorganism belongs to the moderate producers of biofilms since the optical density of the sample (density of the sample - Ds) exceeded the optical density of control (density of the control - Dc) by 3 times. In Y. enterocolitica R-form (D ≤ 0.197) weakly produced biofilms, the optical density of the sample exceeded the optical density of the control by <2 times. Conclusion: The ability to form biofilms, the variability of phenotypic features, and the multiplicity of virulence factors of bacteria significantly reduce the effectiveness of diagnostic studies. The development of accelerated methods of detection and differentiation of the virulent properties of pathogenic bacteria will allow scientifically to substantiate and develop a set of measures aimed at preventing animal diseases and obtaining safe livestock products to prevent human diseases. Thus, we need to pay attention to which forms of colonies do Y. enterocolitica form on solid nutrient media: S- or R-forms. Through this study, we know that bacteria-forming S-shaped colonies are more capable of forming biofilms than R-forms. It means that they are more pathogenic and can cause persistent infections due to adhesion and biofilm formation.

Histological and Immunohistochemical Effects of Neuroectodermal Cells Transplantation after Spinal Cord Injury in Rats

Background: In spinal cord injury, radical treatment is still a persistent hope for patients and clinicians. Our study aimed to determine the different histological changes in central, cranial and caudal sites of compressed spinal cord as a result of neuroectodermal stem cells (NESCs) transplantation in rats. Material and methods: For extraction of NESCs, future brains were extracted from mice embryos (10-days old) and cultured.&nbsp; Eighty, male rats were divided randomly into control, sham (20 rats each); while 40 rats were subjected to compressed spinal cord injury (CSCI). Seven days after spinal cord injury, rats were subdivided into 2 groups (20 rats each); an untreated and treated with NESCs injected cranial and caudal to the site of the spinal cord injury. Rats were sacrificed 4 weeks after transplantations of NESCs and specimens from the spinal cord at the central, cranial and caudal to site of spinal cord injury were proceeded to be stained with haematoxylin &amp; eosin, osmic acid and Immunohistochemistry of glial fibrillary acidic protein (GFAP). Results: Sections of CSCI revealed areas of hemorrhages, necrosis and cavitation limited by reactive astrocytosis, with upregulation of GFAP expression. Evidence of remyelination and mitigation of histopathological features, reactive astrocytosis in CSCI sections were more pronounced in cranial than in caudal region. Conclusions: NESCs transplantation ameliorated the pathological changes, promoted remyelination.

METHODOLOGICAL APPROACHES TO THE STUDY OF FORMATION OF BIOFILMS BY CONDITIONALLY PATHOGENIC AND PATHOGENIC ENTEROSBACTERIES

To study the process of biofilm formation, microorganisms were cultured in 96-well plates, on meat-peptone broth, stained with a 0,1% solution of crystalline violet for 10...15 minutes, after which the unbound dye was washed off. The quantitative accounting of the bound dye was carried out by spectrophotometry at a wavelength of 490 nm. The technique for making bacterial preparations for light and scanning electron microscopy on dodged glasses immersed in Petri dishes with a liquid nutrient medium is proposed. A suspension of bacteria at a concentration of 105 m.k/ml in a volume of 5 ml was shaken on Vortex apparatus and introduced into Petri dishes with 20 ml of meat-peptone broth. Sterile non-greased cover glasses were placed on sterile object glasses and immersed in a liquid nutrient medium in Petri dishes. The material was incubated for 18...24 hours at 37 °C. Then the cover slips were removed with tweezers and some of them were stained with 1% aqueous solution of methylene blue (for light microscopy), and some were placed in Petri dishes with bottomed filters (for electron microscopy). The latter, in order to preserve natural architectonics, were fixed in vivo by pairs of 25% glutaraldehyde for 3...5 hours. Vapors of 2...4% osmic acid solution were used for 2...3-minutes to contrast the preparations. After treatment with vapors of osmic acid, biofilms with included bacteria acquired yellowish or brown color. The obtained preparations after dehydration with propylene oxide vapors and spraying with gold ions were examined in a scanning electron microscope (SEM). The technique allows us to study the phases of development of biofilms and obtain objective data on the morphology of populations of pathogenic and conditionally pathogenic bacteria without disturbing natural architectonics. It is shown that the intensity of biofilm formation by pathogenic microorganisms, such as salmonella, Yersinia, Staphylococcus aureus was slightly higher than that of non-pathogenic: Escherichia, Proteus, Citrobacter, Enterobacter.

EXPERIMENTAL STUDY OF ANTIBACTERIAL DRUGS ON PSEUDOMONAS AERUGINOSA BIOFILMS

The article presents the results of stages of biofilm formation and sensitivity to antibacterial drugs of Pseudomonas aeruginosa bacteria. The technique for preparation of drugs for investigation in an optical and scanning electron microscope has been developed, which allows one to consistently study populations of Pseudomonas aeruginosa bacteria without disturbing natural architectonics. The culture of gram-negative bacteria Pseudomonas aeruginosa №17 in the logarithmic growth phase of growth was used in the work. A solution of canned bile of cattle and pigs, antibiotics of different groups and disinfectants were used as antibacterial preparations. To study the morphology of the P. aeruginosa population, the preparations were fixed in vapor of 25% (by DW) solution of glutaraldehyde. For staining, pairs of a 1% aqueous solution of osmic acid (OSO4) were used, dehydrated with propylene oxide vapor. The action of antibacterial drugs of various groups were studied in the population of P. aeruginosa. Studying the sensitivity of bacteria by diffusion to agar using standard commercial discs, it was found that P. aeruginosa is sensitive to the β-lactam antibiotic group, aminoglycosides, quinolones. Experimental data on the study of the effect of antibacterial drugs on processes of biofilm formation expand the boundaries of knowledge in the field of reseaches of the adaptive capabilities of ubiquitous bacteria for long persistence in both the warm-blooded organism and in environmental objects.

REGULARITIES OF DEVELOPMENT OF BIOFILM OF BACTERIA AT THE NATIVE PHASES OF THEIR FORMATION IN VITRO

The objects of research were the bacterial cultures of the genera Salmonella, Escherichia, Pseudomonas, Staphylococcus. The morphology of biofilms and the phases of its development were studied by the method of light and scanning electron microscopy. To study the formation of biofilms in liquid nutrient media, 24-hour microorganism cultures in S-form grown on dense or liquid nutrient media were used. The material was incubated for 24 hours at 37 °C. Then the cover glasses with biofilms formed on them were extracted with tweezers and placed in Petri dishes with filters laid on the bottom (for electron microscopy). In order to preserve the natural architectonics, were fixed by vapor of 25% glutaraldehyde during 3...5 hours. For contrasting preparations, vapor of 2...4% solution of osmic acid were used for 2...3 minutes. The laws of biofilm formation in conditionally pathogenic and pathogenic bacteria were experimentally confirmed: adhesion of single cells, association of exopolysaccharide matrix into clusters, formation of mature biofilm. SEM method shows the possibility of forming a multilayer structure of biofilm, which determines their increased resistance to antimicrobial agents (antibiotics, disinfectants).

5 PARTHENOGENETIC EMBRYONIC STEM CELLS ARE CONNECTED BY FUNCTIONAL INTERCELLULAR BRIDGES

We previously reported that parthenogenetic stem cells display abnormal centrosome and spindle formation that results in severe chromosome missegregation, with a high incidence of hypoploid karyotypes. Unexpectedly, this is not accompanied by a correspondingly high rate of apoptosis and, by contrast, parthenogenetic cells share the pluripotency, self-renewal and in vitro differentiation properties of their bi-parental counterparts. We hypothesise that this is possible through a series of adaptive mechanisms that include the presence of intercellular bridges similar to those that connect germ cells during spermatogenesis. This would provide a way for mutual exchange of missing cell products, thus alleviating the unbalanced chromosome distribution that would otherwise hamper normal cell functions. The presence of intercellular bridges was investigated in pig parthenogenetic embryonic stem cells (PESC) by transmission electron microscopy (TEM). Cultured cells were fixed in 2% glutaraldehyde and post-fixed in 1% osmic acid. After standard dehydration in ethanol series, samples were embedded in an Epon-Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica). Thin sections were stained and observed with a Jeol 1010 electron microscope. Pig PESC were also subjected to scanning electron microscopy (SEM). To this purpose, they were fixed and dehydrated as described above, covered with a 9-nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech) and examined with an SEM-FEG Philips XL-30 microscope. To demonstrate functional trafficking activity through intercellular canals, fluorescent 10-kDa dextran was injected into the cytoplasm of a single cell with FemtoJet Microinjector (Eppendorf). Movement of the molecule from the injected cell to others was observed with a Nikon Eclipse TE200 microscope. Ultra-structural analysis of PESC demonstrated the existence of intercellular bridges that ensured cytoplasmic continuity among cells. These canals appeared variable in size and were characterised by the presence of stabilising actin patches. Furthermore, extensive movement of 10-kDa dextran among cells demonstrated functional intercellular trafficking through these communication canals, suggesting their use for transfer of mRNA, proteins and ribosomes among cells. Our results demonstrate that PESC present a wide network of functional intercellular bridges that may constitute an adaptive mechanism to support normal cell functions. This process is commonly observed in transformed cells and gives further support to the recent hypothesis that suggests the existence of common features and links between oncogenesis and self-renewal in pluripotent cell lines. Supported by AIRC IG 10376. PG was supported by INGM.