Prevention of excessive exercise-induced adverse effects in rats with Bacillus subtilis BSB3.

AIMS: To characterize efficacy of the Bacillus subtilis BSB3 (BSB3) strain in the prevention of excessive exercise-induced side effects and in maintaining stability of the gut microbiota. METHODS AND RESULTS: Rats were pretreated by oral gavage with B. subtilis BSB3 (BSB3) or with phosphate-buffered saline (PBS) twice a day for 2 days, and were either exposed forced treadmill running or remained sedentary. Histological analysis of intestine, immunofluorescence staining of tight junction (TJ) proteins, serum lipopolysaccharide and intestinal fatty acid-binding protein assay, culture-based analysis and pyrosequencing for the gut microbiota were performed for each rat. Forced running resulted in a substantial decrease in intestinal villi height and total mucosa thickness, the depletion of Paneth cells, an inhibition of TJ proteins expression. Short-term treatment of rats with BSB3 before running prevented these adverse effects. Culture-based analysis of the gut microbiota revealed significant elevation of pathogenic microorganisms only in treadmill-exercised rats pretreated with PBS. High-throughput 16S rRNA gene sequencing also revealed an increase in pathobionts in this group. Preventive treatment of animals with BSB3 resulted in predominance of beneficial bacteria. CONCLUSIONS: BSB3 prevents excessive exercise-associated complications by beneficial modulation of the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study shows a new application of beneficial bacteria for prevention the adverse effects of excessive exercise.

Yeast fermentate prebiotic improves intestinal barrier integrity during heat stress by modulation of the gut microbiota in rats.

AIMS: To evaluate efficacy of Saccharomyces cerevisiae fermentate prebiotic (EH) in protection of intestinal barrier integrity in rats during heat stress, to analyze the impact of heat stress and preventive treatment with EH on the structure of the gut microbiota. METHODS AND RESULTS: Two groups of rats were treated orally with EH or phosphate-buffered saline for 14 days. On day 15, half of the rats in each group were exposed to heat stress conditions, while control animals were kept at room temperature. Histological and Western blot analyses of the intestine, culture-based microbiological analysis and high-throughput 16S rRNA sequencing for the gut microbiota were performed for each rat. Exposure of animals to heat stress conditions resulted in inhibition of tight junction (TJ) proteins expression, decrease of Paneth and goblet cells, decrease of beneficial and increase of pathogenic bacteria. Oral treatment of rats with EH before stress significantly prevents these adverse effects by elevation of the gut beneficial bacteria, particularly butyrate-producing bacteria. CONCLUSIONS: Essential effect of EH in protection of intestinal barrier integrity during heat stress is connected with beneficial modulation of the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results will contribute to the development of new approaches to prevention of heat stress-related complications.

Oral administration of Bacillus subtilis strain BSB3 can prevent heat stress-related adverse effects in rats.

AIMS: To determine the efficacy of Bacillus subtilis strain in prevention of heat stress-related complications in rats. METHODS AND RESULTS: Male Sprague-Dawley rats weighing 250-300 g were treated by oral gavage with B. subtilis BSB3 strain or PBS twice a day for 2 days. Half of the rats of each group were exposed to heat stress (45°C, relative humidity 55% for 25 min), while the remaining rats were placed at identical conditions but at 25°C. Bacterial translocation, histological changes in the intestine, cytokines profile, serum lipopolysaccharide level (LPS), as well as vesiculation of erythrocytes were analysed and compared between groups. Adverse effects of heat stress (morphological changes in intestine, bacterial translocation, elevated levels of LPS and IL-10, increased vesiculation of erythrocytes) were observed only in rats not protected with B. subtilis strain and exposed to heat. All registered parameters in rats pretreated with bacilli and exposed to heat were similar to control groups. CONCLUSIONS: Bacillus subtilis BS3B strain was effective in the prevention of complications related to heat stress in rats. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute towards better understanding of probiotics' mechanisms of action and will bring new approaches to characterize and use of beneficial bacteria.

Diversity and censoring of landscape phage libraries.

Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd-tet (landscape libraries). They were obtained by replacement of amino acids 2-4 and 2-5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space.

[A study of the safety of probiotic bacilli].

The aim of the study was to evaluate the biological safety of a range of probiotic bacilli, ingredients of the commercial preparations biosporin, bactisubtil, and irilis; their biological effects were scrutinized in an animal experiment. The study demonstrated that the strains B. subtilis 3, 07, and B. licheniformis 31, 09, ingredients of biosporin and irilis, unlike B. cereus IP 5832, an ingredient of bactisubtil, were not toxic, toxigenic, or virulent, and did not impair the antitoxic hepatic function, which allows considering them to be safe.

[Influence of probiotic subalin on efficiency of antitumor vaccine].

Preparation subalin created on the basis of a recombinant strain of B. subtilis 2335/105 containing the gene of synthesis of human a-2-interferon has been tested in the experiment for its ability of increasing the efficiency of antitumor vaccine prepared from syngenic tumor cells and cytotoxic lectin--the metabolism product of the strain of B. subtilis B-7025. On the models of Lewis lung carcinoma of C57B1 mice and sarcoma-37 of Balb/c mice it was shown that the complex use of the antitumor vaccine and subalin makes for the more efficient tumor growth suppression and survival of treated animals as compared with the separate use of autovaccine or subalin. The prospects and expediency of the complex use of vaccine and subalin at malignant tumors immunotherapy were established.

[Pre-clinical trials of new spore probiotics].

The paper presents the results of a pre-clinical trial of Bacillus bacterial strains that are ingredients of biosporin, bisporin, and irilis, new Russian probiotic preparations. The study included determination of the cultural and biochemical properties of the probiotic strains, their antagonistic activity, adhesion ability, resistance to various biological liquids, and the length of their persistence in mouse alimentary tract. The listed properties were compared with those of foreign commercial preparations, bactisubtil and enterogermin. The study demonstrated high antagonistic activity of biosporin, bisporin, and irilis against many bacteria of different taxonomic groups, and Candidae. Besides this, the study showed that, unlike similar foreign preparations, the Russian ones were characterized by relatively low adhesive ability, short-term persistence, and absence of virulence factors. According to the results of the study, the most prospective probiotic strains for further clinical application are B. subtilis VKPM B-8611 and B. licheniformis VKPM B-8610, an irilis ingredient.

[Development of nutrient medium for the cultivation of Lactobacillus plantarum 8R-A3 ]. / Razrabotka pitatel'noi sredy dlia kultivirovaniia Lactobacillus plantarum 8R-A3.

Optimal nutrient medium providing accumulation of cell biomass of Lactobacillus plantarum 8R-A3 has been developed. According to this index the proposed medium meets the nutrition requirements of bacteria under study and is comparable with the basal (MRS) medium. Dependence of growth properties of Lactobacillus plantarum 8R-A3 on concentration of the medium basal components has been studied. Maize extract (6 vol.%) and enzymolysate of fodder yeast (5 vol.%) are the most favourable sources of nitrogen nutrition. Addition of pantothenic, nicotinic acids or riboflavin to the nutrient medium increased the yield of Lactobacillus plantarum biomass.

[Spore probiotics]. / Sporovye probiotiki.

Data on probiotic preparations prepared from spore-forming bacteria is reviewed. Information on indications for use, effectiveness and the mechanism of action is given. Preparations being produced in Russia (Biosporin, Sporobacterin and Bactosporin) are evaluated in detail as well as the prospects of the application of probiotics from spore-forming bacteria.

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread.

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.

[Probiotics based on live cultures of microorganisms]. / Probiotiki na osnove zhivykh kul'tur mikroorganizmov.

The modern state of probiotic design and production was discussed in the survey. The worldwide data concerning types of probiotics and their use for restoration of resident microflora of hot-blooded animals and people were systematized. Much attention has been recently paid to the use of the natural preparations to maintain and regenerate the state of the resident microflora of animals and people. These preparations are known as probiotics. The term "probiotic" means microorganisms or substances which are capable to render sanitary effect on macroorganism. I. I. Mechnikov's concept on detoxifications of harmful substances formed in the digestive tract of men by probiotics is expounded. The modern concepts of the probiotics division into groups have been presented. Different kinds of industrial probiotics were considered. The mechanism of positive action of probiotics and their mutual relations with micro- and macroorganisms were provided. The scientific substantiation of new probiotic design was presented.

In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics.

A limited number of antibiotics can be used against Helicobacter pylori infection, and resistance jeopardizes the success of treatment. Therefore, a search for new agents is warranted. The use of probiotics to enhance gastrointestinal health has been proposed for many years, but the scientific basis of the prophylactic and therapeutic actions of probiotics has not yet been clearly delineated. Probiotic strain Bacillus subtilis 3, whose safety has previously been demonstrated, is known to have antagonistic properties against species of the family Enterobacteriaceae. In the present study, it was also found to inhibit H. pylori. The anti-H. pylori activity present in the cell-free supernatant was not related to pH or organic acid concentration. It was heat stable and protease insensitive. At least two antibiotics, detected by thin-layer chromatography (R(f) values, 0.47 and 0.85, respectively) and confirmed by high-performance liquid chromatographic analysis, were found to be responsible for this anti-H. pylori activity. All H. pylori strains tested were sensitive to both compounds. One of these compounds was identified as amicoumacin A, an antibiotic with anti-inflammatory properties. MICs for H. pylori determined in solid and liquid media ranged between 1.7 and 6.8 microg/ml and 0.75 and 2.5 microg/ml, respectively. The underestimation of MICs determined in solid medium may be due to physicochemical instability of the antibiotic under these test conditions. An additive effect between amicoumacin A and the nonamicoumacin antibiotic against H. pylori was demonstrated.

[Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain]. / Eksperimental'naia otsenka biobezopasnosti genno-inzhenernykh bakterii na modeli shtamma Bacillus subtilis, produtsiruiushchego interferon.

The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B. subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model. As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants. The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

Simplified technique for identification of the aerobic spore-forming bacteria by phenotype.

The use of modern research approaches of genetics, biochemistry and molecular biology has led to progress in bacterial taxonomy. Systematic study of the aerobic spore-forming bacteria has resulted in the realignment of the genus Bacillus into several new genera. In the meantime, the identification process has become more difficult for the non-specialist in Bacillus taxonomy. This paper presents a key for the simplified phenotypic identification of the mesophilic, aerobic, spore-forming bacteria belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Geobacillus and Virgibacillus. A total of 81 species were included and 115 morphological and physiological tests were analysed for their discriminative efficiency. This key is practical for rough but quick identification of aerobic spore-forming bacteria isolated from nature. Such preliminary identification will be helpful for the selection of reference strains and methods for more precise identification using the newest techniques. The reliability of the proposed identification key was tested on 100 cultures from the Ukrainian Collection of Microorganisms. The developed identification key is represented in interactive mode on a website (http://www/imv.kiev.ua/key/).

[Bacteria of Bacillus species--prospective source for biologically active substances]. / Bakterii roda Bacillus--perspektivnyi istochnik biologicheski aktivnykh veshchestv.

The present data from literature on bio-active substances produced by aerobic sporulating bacteria of Bacillus genus are presented. Problems of antibiotics synthesis by bacilli are considered. The antibiotics under consideration are both well-known and new ones--biosurfactants characterized by the surface-active properties. Enzymic activity of Bacillus genus bacteria has been characterized. Special attention was given to the analysis of enzymes, which could play a positive part in the digestion processes. Data are presented which evidence that the bacilli produce the substances, which affect essentially the immune reactivity of the macroorganism.

[The effect of antibiotic preparations and their combinations with probiotics on the intestinal microflora of mice]. / Vplyv antybiotychnykh preparativ ta ïkh kombinatsii z probiotykamy na mikrofloru kyshechnyka myshei.

Antibiotic drugs (biseptol, polymyxin, canamycin) as well as their combinations with probiotics biosporin and subalin have been studied for their effect on mice intestine microflora. It has been established that under peroral administration of the above drugs dysbacteriosis of the 2-3rd degrees arises in animals; under their combined use with probiotics one can prevent the development of dysbacteriosis to considerable extent. It has been shown that dysbacteriosis of the 2-3rd degrees can be liquidated for 5-7 days, using probiotics biosporin and subalin. No self-recovery of normal intestinal microbiocenosis is observed in this period. Inconsiderable variations in the composition of normoflora under combined use of antibiotics with probiotics are renewed on the 3-5th day after stopping the drugs intake.

[The identification of freshly isolated strains, the causative agents of human candidiasis and the search for effective antifungal probiotics]. / Identyfikatsiia svizhovydilenykh shtamiv--zbudnykiv kandydoziv liudyny i poshuk efektyvnykh protyhrybkovykh probiotykiv.

The species variety of yeasts of the genus Candida in children and patients with AIDS has been investigated. Composition of the studied species was found to be similar in newborns and in women with candidiasis. In order to create new probiotics with antifungal properties, the cluster analysis of antagonistic activity of some Bacillus strains against Enterobacteriaceae, Candida, normoflora and food-fermenting lactobacteria has been conducted. There were selected the combinations of some Bacillus strains, which effectively inhibited pathogenic and opportunistic microorganisms and at the same time had no effect on intestinal normoflora.

[The monosaccharide composition of the glycocalyx in Mollicutes and phylogenetically related bacteria in the genus Bacillus]. / Monosakharydnyi sklad hlikokaliksu deiakykh molikutiv i filohenetychno blyz'kykh ïm bakterii rodu Bacillus.

A number of monosaccharides composing the glycocalix carbohydrates of some Mollicutes (Mycoplasma pneumoniae FH, M. hominis PG21, M. fermentans PG18, Acholeplasma laidlawii PG8, A. laidlawii var. granulum st. 118) and phylogenetically related bacteria from the genus Bacillus (Bacillus cereus 89, B. cereus DM423, B. subtilis 1/2, B. licheniformis 31, B. licheniformis 49) were detected using plant lectins of definite carbohydrate specificity labelled by colloid gold. The structure of extracellular glycopolymers of microorganisms was discussed concerning their role in the adhesion process and their specializations in colonization of the appropriate organs and tissues of macroorganisms as well as the action of probiotics on the basis of bacteria from the genus Bacillus. It was marked, that the mollicute cells typically interacted with all vegetable lectins tested while bacilli studied bind certain lectins only. The surface carbohydrates prevailing in monosaccharides from tested one for superficial structures of Bacillus pathogenic strains were: sialic acid, beta-D-galactose and alpha-N-acetyl-D-galactosamine, while L-fucose and alpha-D-glucose were observed in insignificant quantity or were absent. The nonpathogenic Bacillus strains being a basis of biopreparations were distinguished by the insignificant amount of sialic acid and other monosaccharides in the composition of carbohydrates of their glycocalix. Thus, as a result of research it was confirmed, that the superficial glycopolymers of Mollicutes and some related bacilli were alike as to presence of some monosaccharides that depended on taxonomic position of microorganisms and their biological activity.

[The effect of the cultivation conditions on the properties of bacilli comprising the basis of probiotics]. / Vliianie uslovii kul'tivirovaniia na nekotorye svoistva batsill, sostavliaiushchikh osnovu probiotikov.

Such biological factors as bile, gastric juice, blood serum, amino acids and pH of the medium have been studied for their effect on the growth intensity and antagonistic activity of bacilli being the basis of biosporin and subalin. It has been established that pH of the medium within 7.0-9.0 as well as certain concentrations of the gastric juice, bile, blood serum and most of amino acids did not affect the growth of Bacillus subtilis 3 and Bacillus subtilis 2335/105. A regular decrease of growth intensity in bacilli under the increase of the gastric juice concentration or decrease of pH of the medium to 5.0-2.0 is registered. The mentioned biological factors affect differently the antagonistic properties of the studied cultures.

[The safety and reactogenicity of the new probiotic subalin for volunteers]. / Izuchenie bezopasnosti i reaktogennosti novogo probiotika subalina dlia dobrovol'tsev.

Data are presented on the study of innocuousness and reactogenicity of a new recombinant probiotic subalin characterized by antibacterial and antiviral activity. Volunteers who took subalin (one dose twice a day for 10 days) were under observation at Kyiv Scientific-Research Institute of Epidemiology and Infectious Diseases of the Ministry of Public Health of Ukraine. They carried out clinical and bacteriological investigations during administration and after termination of the course of oral administration of subalin testified to its innocuousness as well as to the absence of any side-effects.