Effect Of Cell-free Extracts Research Articles

ADHESIVE PROPERTIES OF BACTERIA UNDER THE INFLUENCE OF BIFIDOBACTERIUM BIFIDUM 1 AND LACTOBACILLUS REUTERI DSM 17938I CELL-FREE EXTRACTS

Increasing in the adhesive potential of probiotic bacteria on the one hand and weakening the adhesive ability of the pathobionts on the other is a pathogenetically based approach in the fight against intestinal infectious diseases and intestinal dysbiosis. At present postbiotic products are considered as one of the most promising means of influencing the intestinal microbiota. The aim of the work was to study the effect of cell-free extracts containing derivatives of Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 on the adhesive properties of probiotic (L. reuteri DSM 17938 and B. bifidum 1) and opportunistic bacteria (Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922). Four cell-free extracts were investigated: L – obtained from the L. reuteri disintegrate; ML – obtained from the L. reuteri culture, cultivated in its own disintegrate; B – obtained from the B. bifidum disintegrate; MB – obtained from the B. bifidum culture, cultivated in its own disintegrate. The effect of cell-free extracts on the adhesive properties of microorganisms was studied by using Brilis method. ML, MB, and B extracts were found to be able to significantly increase the adhesive potential of B. bifidum and L. reuteri. Under their influence adhesion index calculated for bifidobacteria increased by 41.1 % (ML), 41.5 % (MB) and 50.6 % (B), and the microorganism adhesion index calculated for lactobacilli increased by 30.77 % (ML), 44.45 % (MB) and 44.97 % (B). Both studied probiotic bacteria moved up from the category of «moderately adhesive» into the category of «strongly adhesive» with microorganism adhesion index > 4.0. Lactobacilli also became «strongly adhesive» under the influence of L extract, while microorganism adhesion index increased by 37.9 %. Among the studied extracts, only L extract caused a significant increase in the adhesion index of the S. aureus test culture. The other extracts caused minor changes in the adhesive potential of the test cultures. Under their influence, there was a tendency to increase or decrease the adhesive ability of bacteria. Under the influence of MB extract, the S. aureus test culture passed from the «strongly adhesive» category to the «moderately adhesive» category, and the E. coli test culture transferred from the «moderately adhesive» category to the «weakly adhesive» category. The data obtained are of great importance when developing new metabiotics based on the B. bifidum 1 and L reuteri DSM 17938 probiotic strains derivatives.

APPLICATION EFFICIENCY OF BIFIDOBACTERIUM BIFIDUM 1 AND LACTOBACILLUS REUTERI DSM 17938 CELL-FREE EXTRACTS IN VIVO

Insufficient efficiency and safety of cellular probiotics encourages the search for new effective means of correction of microecological disorders. Most of the beneficial effects of probiotics are due to the biological activity of their structural components and metabolites. Recently, great hope is pinned on postbiotic products as a means of restoring the balance of intestinal microbial populations. The data obtained in this experimental study demonstrate the ability of cell-free extracts from Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 cultures, cultivated in their own disintegrates supplemented with ascorbic acid, to provide anti-infection protection and correct microecological disturbances at modeling an infectious process against a background of antibiotic-induced dysbiosis in mice. The beneficial effects of cell-free extracts showed up in the acceleration of the pathogen elimination and an increase in the number of representatives of the positive intestinal microbiota. The results of the study justify the need for further clinical trials to determine the therapeutic efficacy of cell-free extracts when included in the protocols of dysbiosis treatment.

Staphylococcal proliferation and biofilm formation in vitro under the influence of cell-free extracts of probiotic origin

The paper presents the results of the study on proliferation and biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis under the influence of cell-free extracts obtained by the author’s method and containing derivatives of probiotic strains Вifidobacterium bifidum and Lactobacillus reuteri.The aim of this work was to study the ability of cell-free extracts containing derivatives of probiotics Bifidobacterium bifidum and Lactobacillus reuteri to influence proliferation and biofilm formation by staphylococci in vitro, to evaluate the prospects of their use for the correction of microecological disorders and adjuvant therapy of staphylococcal infection.Materials and methods. Cell-free extracts were obtained from commercial strains B. bifidum and L. reuteri by the authors’ method. Reference strain of S. aureus AТСС 25923 and clinical isolate of S. epidermidis were used as a test cultures. The investigation of the proliferation and biofilm formation by staphylococci was carried out by spectrophotometric method using a microtiter-plate reader “Lisa Scan EM” (Erba Lachema s.r.o., Czech Republic).Results. It has been established that the effect of cell-free extract on proliferation and biofilm formation depends on the type of extract and on the species of staphylococcus. Among the five studied extracts, only one significantly inhibits the proliferation and biofilm formation of both staphylococci species. It is the cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose. The proliferative activity of S. aureus is sensitive to the L. reuteri derivatives while the proliferative activity of S. epidermidis is sensitive to the B. bifidum derivatives. The filtrates of disintegrates have stimulatory effect, while the filtrates of cultures have inhibitory effect on the staphylococcal proliferation. The biofilm formation by S. aureus is significantly inhibited by B. bifidum derivatives and is stimulated by L. reuteri derivatives. The biofilm formation by S. epidermidis is stimulated by derivatives of bifidobacteria and does not change in the presence of derivatives of lactobacteria in the growth medium.Conclusions. Obtained results indicate a high bioregulatory potential of cell-free extracts of probiotic origin and the possibility of drugs development for microecological disorders correction on their basis. They also confirm that the method of obtaining probiotic derivatives with bacteriotropic activity through precursor-directed biosynthesis is promising. Cell-free extract, obtained from L. reuteri culture, grown in its own disintegrate supplemented with glycerol and glucose, exhibits pronounced anti-staphylococcal activity in vitro. After confirming efficacy in vivo, it can be recommended for the adjuvant therapy of staphylococcal infections.

Bifidogenic properties of cell-free extracts derived from probiotic strains of Bifidobacterium bifidum and Lactobacillus reuteri

Comprehensive study of the biological activity of structural components and metabolites of “beneficial” microorganisms opens the prospects of efficient and rational use of their biotechnological potential in the correction of microecological and related disorders. The study tested proliferative activity and biofilm formation by Bifidobacterium bifidum probiotic strain under the influence of cell-free extracts containing structural components and metabolites of the probiotic strains of B. bifidum and Lactobacillus reuteri. Cell-free extracts were obtained by disintegrating suspensions of probiotic cells by cyclic freezing-thawing, cultivating probiotic microorganisms in their own disintegrates and subsequent filtration of the obtained disintegrates and cultures. The proliferative activity and biofilm formation of the probiotic test culture were studied by spectrophotometric microtiter plate method with 10%vol, 30%vol and 50%vol content of cell-free extracts in the cultivation medium. All investigated extracts showed a significant concentration-dependent stimulatory effect on the proliferative activity of B. bifidum. According to the degree of stimulatory effect on the B. bifidum proliferation, cell-free extracts arranged in ascending order: MLG (filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose) < MB (filtrate of В. bifidum culture, grown in В. bifidum disintegrate) < B (filtrate of В. bifidum disintegrate) < ML (filtrate of L. reuteri culture, grown in L. reuteri disintegrate) < L (filtrate of L. reuteri disintegrate). With the same content in the culture medium, filtrates of disintegrates had a more pronounced stimulatory effect than filtrates of cultures grown in their own disintegrates. Cell-free extracts from L. reuteri (L and ML) exerted a more pronounced stimulatory effect than cell-free extracts from B. bifidum. Not all studied cell-free extracts stimulated the biofilm formation by B. bifidum. The effect of cell-free extracts on this process depended on their type and concentration. Extract L had a predominantly inhibitory effect on biofilm formation by B. bifidum. The most pronounced stimulatory effect on biofilm formation by B. bifidum came from extract MLG. ML, B and MB extracts stimulated this process approximately equally. The detection of significant bifidogenic effect of the studied cell-free extracts may contribute to their pharmaceutical applications. Cell-free extracts can be used as metabiotics or prebiotics for increasing the survival of the injected probiotic, facilitating its inoculation in the gastrointestinal tract when used together. The obtained data encourage further careful study of the biochemical composition of cell-free extracts and efforts to clarify the mechanism of their action.

Combined effects of plant and cell-free extracts of lactic acid bacteria on biogenic amines and bacterial load of fermented sardine stored at 3 ± 1 °C

Effects of cell-free extracts (CFE, 8%) prepared using Lactobacillus plantarum FI8595 or Pediococcus acidolactici ATCC 25741 alone or in combination with thyme and laurel extracts (0.5%) on physical, sensory, microbiological and chemical quality of fermented and vacuum packaged sardine fillets were investigated during 8 wk of chilled storage (3 ± 1 °C). Sardine fillets were divided into 7 groups with the control being untreated. The other groups were treated with CFE of Lb. plantarum (LP group) and P. acidolactici (PA group). In addition, thyme with LP CFE (THLP) or PA CFE (THPA); and laurel with LP CFE (LALP) or PA CFE (LAPA) were also prepared. According to the sensory and physical assessment, shelf life of fermented fish was 5 wk for the control, 6 wk for LP, PA, LALP and LAPA, and 7 wk for THPA and THLP, although chemical and microbiological results exhibited longer shelf life. THPA and THLP had the lowest bacterial growth and TBA values. TVB-N values remained below 35 mg/100 g until 7 wk for all groups. THPA significantly reduced TMA formation. The study suggested that cell-free extracts of Lb. plantarum and P. acidolactici with thyme extract was the most effective way to improve the shelf-life of fermented sardines stored at chilled temperature.

Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells.

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell’s fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs’ viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.

THE IMPACT OF PROBIOTIC BACTERIUM Lactobacillus acidophilus IN GROWTH AND SURVIVAL OF THE JUVENILE FRESH WATER RIVER PRAWN (Macrobrachium rosenbergii) INFECTED WITH PATHOGENIC Vibrio spp.

The present study was performed to investigate the aptitude of Lactobacillus acidophilus 04 isolated from home made curd as a probiotic in treating the vibriosis of freshwater river prawn (Macrobrachium rosenbergii). Both in vivo and in vitro analyses were performed to test their efficacy. The antagonistic effect of cell free extract obtained from L. acidophilus 04 biomass was tested against three pathogenic Vibrio spp. namely Vibrio harveyi, V. vulnificus and V. anguillarum using disk diffusion method. The result showed inhibitory effects of cell-free extract on all three Vibrio spp. A feeding experiment was conducted for a period of 30 days by incorporating L. acidophilus in shrimp feed in three experimental (T1,T2, T3) and one control groups. The result showed an increased gain in size and weight in experimental groups treated with L. acidophilus 04 compared to control group without any L. acidophilus 04 addition. The treatment groups were further challenged in vivo by inoculating shrimps with 104-106 cells of pathogenic Vibrio harveyi, V. vulnificus and V. anguillarum. The histopatholgical and microbiological analysis revealed significant variation (P

Antibacterial Effects of Lactic Acid Bacteria Cell Free Extract on the Growth of Local Isolates of Listeria moncytogenes from some Food Sources

The present study aimed to determine the antibacterial activity of lactic acid bacteria metabolites against Listeria monocytogenes which was isolated from some foods in local markets of Erbil and Koya cities; 225 food samples were examined for the presence of Listeria species particularly L. monocytogenes from October 2010 to March 2011. The studied samples included 70 samples of raw chicken meat, 55 samples of raw milk, 50 samples of cheese and 50 samples of raw red meat. According to motility test, hemolysin production, sugar fermentation test, 8 isolates of L. monocytogenes were identified, 1 (2 %) isolate from cheese, 2 (4 %) isolates from red meat and 5 (7.3 %) isolates from chicken meat. The percentages of contaminated foods for Listeria species from chicken meat, raw milk, cheese and red meat were 20, 9, 20 and 22% respectively. With respect to the biocontrolling attempt, the effect of cell free extract (CFE) of lactic acid bacteria (LBA) which is considered as safe against isolated L. monocytogene was used as biocontrolling agent. The CFE of lactic acid bacteria which isolated from local dairy products, was tested for its controlling ability by agar diffusion method, the CFE showed inhibition zone from (15-23 mm) in diameter. Also the minimum inhibitory concentration MIC of CFE was determined at three additive percentages (0.5, 1.0 and 1.5ml to 10 ml of nutrient broth), the inhibition percentage ranged from (28.75 - 48.97) at (0.5 %), (52.13-95.55) at (1 %) and (82.93 -98.15) at (1.5 %) additive percentage of CFE of LAB.

Comparing the effect of probiotic and chlorhexidine as a mouth rinses in bacterial plaque

Probiotic technology represents a breakthrough approach to maintaining oral health by utilizing natural beneficial bacteria commonly found in healthy mouth to provide a natural defense against those bacteria though to be harmful to periodontal tissue. Data are still sparse on the probiotic action in the oral cavity. The aim of study was the present study evaluated clinically the efficacy of probiotic type inarched yeast extract, and chlorhexidine mouth rinses on plaque. Material &method: Four strain of probiotics bacteria were used, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus & bifidubacteriu m actiregularis , and grown in skim milk and plus 0.25, 0.5 % yeast extract individually, the cell free extract of each one of those treatments were add 1: 9 todiluted bacterial plaque taken from 5 subjects, to measure the effect of cell free extract on aerobic & anaerobic bacteria. 45 subjects with chronic periodontitis attend periodontal clinic in college of dentistry university of Baghdad (age 25- 35) the trial design as 14 days comparative study between a probiotic and chlorhexidine. Result: add yeast extract increased the total count of 4 probiotic bacteria 16.25 -29.22% , Cell free extract of Lb. acidophilus probiotic skim milk dairy product was the most powerful in reducing both aerobic and anaerobic plaque bacteria among other probiotic bacteria, it reduced 3.38 - 2.17 log cfu/gr aerobic bacteria while the same treatment reducing the anaerobic bacteria 2.5- 2.1 log cfu/gr , the probiotic and chlorhexidine groups had less plaque accumulations compared with the control group at the end of 14day (P<0.001and P<0.001)respectively . Conclusion: the probiotic mouth rinse was found effective in reducing plague accumulation and gingival inflammation, probiotic mouth rinse obviously has a potential therapeutic value and further study is recommended to determine the efficacy.

A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

Effect of cell-free extracts from Mycobacterium tuberculosis H37Rv on lung succinoxidase.

SEGAL AND BLOCH showed that non-proliferating suspensions of human tubercle bacilli grown in vivo (LRv) exhibited different biochemical properties as compared with the same strain of tubercle bacilli grown in vitro: (a) they had a lower hydrogen transfer capacity; (b) glucose and its intermediates failed to cause an increase over their endogenous respiration1.