Alternative Methods of Evaluation of Antimicrobial Activity of Breast Milk Serum.

Antimicrobial properties are traditionally evaluated by the seeding technique, which is laborious, time-consuming, and rather imprecise. We studied the possibility of using microscopy and spectrophotometry methods for evaluation of cytotoxic activity of breast milk serum against opportunistic microbes. Activity of 50 breast milk samples obtained from healthy women at different lactation stages was tested against opportunistic yeast C. albicans. Microscopy showed that incubation of cell suspension with lactoserum led to destruction of cell walls and cytoplasmic membranes with the formation of vesicular debris that absorbed the dye from the medium. Spectrophotometric measurement of the dye remained in the medium revealed a dose-depended effect of the lactoserum on C. albicans cells and strong inverse correlation between the lactation period and cytotoxic activity of the lactoserum (r=-0.948). These methods can be used in veterinary and food processing for estimation of biological activity of milk.

Silicon-hydroxyapatite‒glycerohydrogel as a promising biomaterial for dental applications.

Nanocomposite silicon-hydroxyapatite‒glycerohydrogel (Si-HA‒glycerohydrogel) with different hydroxyapatite (HA) contents of 0.75 and 1.75 wt.% and the same Si content (2.04 wt.%) was obtained by the sol‒gel method. Silicon tetraglycerolate in the form of glycerol solution was used as a biocompatible precursor and HА in the form of aqueous colloidal suspension - as a template and property modifier. Transmission electron microscopy was applied to demonstrate that there are nanoscale HA particles that are in the crystalline state. For the first time, using the atomic force microscopy method, the remineralizing properties of Si-HA‒glycerohydrogel were studied on human teeth extracted for orthodontic reasons. It was found that Si-HA‒glycerohydrogel containing 1.75 wt.% HA has a pronounced remineralizing effect. Immersion of tooth enamel samples in the gel for one month significantly reduces roughness and makes the enamel surface more uniform. Silicon contained in glycerolates in a biologically active and accessible form exerts an additional positive effect on the process of remineralization of tooth enamel. By the energy dispersive X-ray analysis, it was demonstrated that the tooth enamel had an increased silicon content; and the Vickers microhardness test showed greater microhardness values. The obtained data analysis allows the remineralizing Si-HA‒glycerohydrogel to be considered as a promising biomaterial for dental applications.

[Application of the duplex-specific nuclease for fast analysis of single nucleotide polymorphisms and detection of target DNA in complex PCR products].

We have developed a simple method for fast analysis of single nucleotide polymorphisms and identification of target clones from cloned complex PCR products. The method utilizes Kamchatka crab duplex-specific nuclease and universal fluorescent probe and is alternative to laborious screening procedures using radioactive probes, restriction analysis followed by gel electrophoresis or expensive sequencing. The method efficacy was demonstrated in several model experiments.

[Preparation of prokaryotic cDNA for high-throughput transcriptome analysis].

High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.

[Clinico-laboratory associations of candidial infection persistence in diabetes mellitus].

Population heterogeneity of Candida spp. in feces of patients with diabetes mellitus (DM) demonstrated in our earlier studies prompted to focus on the persistence of candidial infection depending on demographic characteristics of the patients and clinical features of DM. We examined 64 patients with DM and 40 practically healthy subjects aged 20-60 yr. The seeding rate (%) of short-lived (emerged on day 0 or 1) and long-lived (emerged on both days 0 and 1) of Candida spp. cultures was determined. The short-lived fraction of microorganisms most frequently occurred in patients below 50 yr without clinico-laboratory signs of DM and the history of candidiasis (coincidence rate 75.0%, p < 0.001). The long-lived fraction was characteristic of patients with the history of candidiasis having DM of less than 5 yr in duration and glycated hemoglobin over 7.8 mmol/l without complications taking oral hypoglycemic agents (80%, p < 0.001). Candida spp. were totally absent in patients with late diabetic complications, duration of disease over 5 yr, glycated hemoglobin level below 7.8%, and candidiasis-free history. It is concluded that clinico-laboratory characteristics and demographic factors determine persistence of Candida spp. in feces of DM patients.

[Experimental simulation of the structure of gastrointestinal microbial biocenosis].

Structural analysis of human and rat gastrointestinal microbial communities revealed their general similarity. The structure of microbial biocenosis in ileum parietal mucin appears to be highly sensitive to nutritional factors. Inadequate nutrition leads to destruction of microbial microassociations in parietal mucin, a calcium-deficient diet has similar effect in feces. Fiber-rich diets stimulates build-up of indigenous communities while artificial nutrients and calcium-enriched diets promote formation of mixed indigenous-transient microbial associations. Bacteria themselves prove to be weak modifiers of the observed effects.

[Candida and Saccharomyces spp. fungal associations in fecal microbiocenosis of diabetes patients and healthy subjects].

The article is devoted to analysis of pathogenic and diagnostic significance of Candida and Saccharomyces co-existence in diabetic patients. These transient fungi are known to be present in fecal microbiocenosis of both healthy subjects and patients with diabetes mellitus. However, their overall occurrence is significantly increased in the disease and the structure of the biocenosis undergoes alteration. These data confirm the role of yeast-like fungi in pathogenesis of diabetes. The diagnostic value of detection of monospecific and mixed populations of Candida and Saccharomyces spp. is not very high, but their presence in feces, especially in women, may be regarded as a sign of disturbed carbohydrate metabolism.

[Architectonics of gastrointestinal microbial community].

Architectonics of gastrointestinal microbial community in colonic mucosa and feces was studied in patients with different gastrointestinal pathologies and without them. The study model was comprised of Bifidobacteria, Lactobacteria, E.coli, Enterococci, Staphylococci, and Candida. Communities of mucosal and fecal microbes appear to be organized in a definite manner, with most microorganisms forming either indigenous or indigenous-transient associations. Multicomponent indigenous-transient associations prevail in fecal communities, few-component indigenous one or individual microbes in parietal colonic mucin. The structure of community from colonic mucosa overlying a tumour proved identical with that from the apparently normal tissue whereas the community from an inflammed site was structurally closer to the fecal one.

[Colonization of the rat gastrointestinal tract by Staphylococcus aureus biotopes].

Six groups of Wistar rats were fed different diets. Intestinal mural and fecal microflora was analysed. Four types of colonization of trointestinal tract were distinguished after intragastric administration of Staphylococcus aureus depending on a variety of factors affecting composition of microbial communities. Mechanisms of colonization ensuring circulation of S. aureus in the gastrointestinal tract are discussed.

[Influence of various levels of vitamin D and calcium in the diet on variability of microbes association of rats].

It is established, that the microecological system of intestines microbes association structurally reacts to the maintenance of vitamin D and calcium in food. Thus change of frequency of occurrence, the specific maintenance and a dispersion of separate components of system is observed. Besides character and force of correlation communications between concentration of microbes varies. The revealed principles of formation and change of microbic community of intestines enable to define mechanisms of selective management of microbic components microbes association.

[Influence of various dietary intakes on the rat's parietal microflora composition].

It was established that specific and quantitative characteristics of microecosystem of parietal mucin of rat's gastrointestinal tract, significantly varied depending on diet. Microbiocenosis of the colon was the most liable to alimental factor, but in the small intestine it was more depending on the endogenic factors of macroorganism. The principle of forming of microecosystem of parietal mucin of rat's gastrointestinal tract subject to composition of the diet was considered in the article.

[The role of food factor in the forming of the variety of the characteristics of fecal microbiocenosis in rats].

The researchers studied mechanisms of the forming of fecal microbiocenosis in rats on different diets. The composition of nutritional elements had a significant effect on the qualitative and quantitative composition of both indigenous and transitory fecal microflora. Under scant diet, the leading role in the forming of intestinal microbiocenosis belonged to macroorganism characteristics, while under excessive nutrition intestinal microbiocenosis depended mostly on the composition of the dietary intake. The study found that excessive nutrition lowered competition for the nutrition substrate, and indigenous microflora no more prevented the growth of transitory one.

[Influence of different diets on special formation of fecal microbes association].

Aim of the present study was research special formation fecal microbes association depending on different diets. The three groups rats were fed diets to be different on quality and quantity composition. Was established, that diet composition exert influence to frequency situated and contents of all types of fecal microbes. In situation of nutrients deficiency there were not microbes synergism. In situation of increase diet components had been formatting synergetic microbes association, include all of bacteria's types. As result of the present study is the demonstrated possibility nonprobiotic correction dysbiotic condition with different diet's components.

[Influence of Bacillus cereus on microbiocenosis of gastrointestinal tract in rats].

The modifying effect of Bacillus cereus on intestinal microbiocenosis was investigated in eubiotic and disbiotic female rats. Qualitative and quantitative characteristics of gut and mucosal microflora from different parts of rats' intestine were studied before and after intragastral application of B. cereus suspension. The single application of B. cereus suspension resulted in appearance of this bacterium in feces and in parietal mucin from all parts of the intestine. In eubiotic rats compared with disbiotic, B. cereus adhere to parietal mucin much more efficiently and supplanted indigenous microflora. During disbiosis B. cereus sometimes had stimulating effect on the intestinal microbiocenosis. Gut microbiocenosis appeared to be more resistant to B. cereus invasion than mucosal. This fact was considered to be the evidence of higher sensitivity of mucosal microbiocenosis to short-term influence of exogenous microbial factor.

[Microbiocenosis of parietal mucin in gastrointestinal tract of rats with induced disbiosis].

Comparative study of qualitative and quantitative characteristics of parietal mucin microbiocenosis in rats with alimentary induced disbiosis was performed. It has been shown that disbiosis was associated with changes in parietal microflora which were observed mainly in colon mucosa. Disbiosis in parietal area, in contrast to central area manifested by changes in quantitative characteristics of microbiocenosis. Results of the study allowed to suppose that parietal mucosal microbiocenosis was more stable than gut one and that this stability was determined by endogenous homeostatic factors of the host.

[Substrate phase state and time factor in variability of human fecal microbiota].

Effects of substrate phase state and time factor on variability of human fecal microbiota were studied. It was shown that microecological system of native feces was characterized by marked time-dependent variability. It is unstable and begins to destruct after 24 hours of cultivation. The most sensitive elements of the system were bifidobacteria and Escherichia coli. Change of phase state of biotope eliminated the effect of factor limiting the microecosystem development, which allowed species of obligate and transitory microflora to freely colonize the growth substrate and interact with each other. The mentioned facts demonstrate that fecal microbiota exists in the environment of excess of growth substrate, which colonization is limited by cluster structure of biotope of native feces. It was concluded that phase state of growth substrate and duration of cultivation are important factors determining the population variability of fecal microbiota.

[Study of the influence of Staphylococcus aureus on gastrointestinal tract microbiocenosis in rats].

The authors studied the modifying effect of Staphylococcus aureus on the microbial composition of gastrointestinal tract microbiocenosis. The subjects were female rats in the condition of eubiosis or dysbiosis. The species and quantitative composition of the fecal microflora and the parietal mucin in different parts of the intestine were studied after an intragastral administration of St. aureus suspension. A single introduction of St. aureus into the gastrointestinal tract of rats led to the appearance of this microbe in the feces and parietal mucin in all the parts of the intestine regardless the initial condition of the intestinal microbiocenosis. The indigenous microflora, both in eubiotic and dysbiotic conditions, practically did not respond to an intragastral administration of staphylococcus, except a little decrease in the proportion of bifidobacteria. Meanwhile, there was a significant increase in the incidence of candid detection. The indigenous parietal microflora changed more substantially, which demonstrates a higher sensitivity of the parietal microbiocenosis to a short-time exposure to an exogenous microbial factor.

[Comparative evaluation of the safety of poly- and and oligoguanidines].

The antimicrobial activity and microenvironmental safety of poly- and oligoguanidine antiseptics were compared. E. coli and St. aureus test strains were found to be more sensitive to chlorohexidine bigluconate (CHB) upon a long (24 hour) exposure and to polyhexamethylene guanidine derivatives on short (30-second) contact. It is concluded that unlike polyguanidine antiseptics, CHB is more ecologically dangerous; when used for rapid disinfection, it can provoke impairment in the microenvironmental balance outside the area of application. The findings show it expedient to assess the microenvironmental safety of antimicrobial agents in order to prevent ecological catastrophes.

[Comparison of the methods of evaluation of the microbiological danger of antimicrobial agents by the example of oligo- and polyguanidines].

The article contains the results of comparison of the methods of evaluation of the microbiological danger of antimicrobial agents by the example of oligo- and polyguanidines. The study demonstrates that choice of an adequate study model (a bacterial or protozoa species) and exposure time depends on the characteristics of the preparation to be tested. It shows that the microbocide activity of polyguanidines should be studied on a bacterial model using long (24 hours) exposure. The most sensitive to XG6 was a protozoa culture in short-time exposure. To evaluate the biological danger ofantimicrobial agents, it is also possible to apply techniques that allow revealing reversable physiological changes in a culture of test objects.