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

2019 ◽  
Vol 10 (1) ◽  
pp. 124-128 ◽  
Author(s):  
O. V. Knysh
Keyword(s):  
Biofilm Formation ◽  
Proliferative Activity ◽  
Lactobacillus Reuteri ◽  
Probiotic Strain ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Bifidobacterium Bifidum ◽  
Careful Study ◽  
Structural Components ◽  
Probiotic Strains

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.

scholarly journals Influence of cell-free extracts of Bifidobacterium bifidum and Lactobacillus reuteri on proliferation and biofilm formation by Escherichia coli and Pseudomonas aeruginosa

2019 ◽  
Vol 10 (2) ◽  
pp. 251-256 ◽  
Author(s):  
O. V. Knysh ◽  
O. Y. Isayenko ◽  
Y. V. Voyda ◽  
O. O. Kizimenko ◽  
Y. M. Babych
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Proliferative Activity ◽  
Lactobacillus Reuteri ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Bifidobacterium Bifidum ◽  
Plate Method ◽  
E Coli ◽  
Test Cultures

The development of new effective preparations for the correction of microecological disorders based on probiotic derivatives requires a comprehensive study of the biological activity of the latter. We studied the proliferative activity and biofilm formation by clinical isolates: Escherichia coli and Pseudomonas aeruginosa under the influence of cell-free extracts containing structural components and metabolites of the Bifidobacterium bifidum and Lactobacillus reuteri probiotic strains. Cell-free extracts were obtained from disintegrates and cultures of probiotics. Disintegrates were prepared by cyclic freezing-thawing of probiotic cell suspensions. The cultures were obtained by cultivating probiotic microorganisms in their own disintegrates. The obtained disintegrates and cultures were filtered. The proliferative activity of the test cultures was studied using the spectrophotometric microtiter plate method after an hour-long exposure in undiluted cell-free extracts and subsequent cultivation in a nutrient medium containing 30%vol of the studied extracts at 37 °C for 24 hours. The biofilm formation of the test cultures was studied with 30% vol content of cell-free extracts in the cultivation medium using the spectrophotometric microtiter plate method. All the studied extracts exerted a similar effect on the proliferative activity and biofilm formation by E. coli and P. aeruginosa. Exposure of the test cultures in all undiluted extracts during an hour led to a significant decrease in the optical density of the test samples: optical density of the test wells ranged from 36.5% to 49.8% of the control wells. The test cultures that were exposed to the extracts: filtrate of L. reuteri disintegrate (L), filtrate of В. bifidum disintegrate (B) and filtrate of В. bifidum culture, grown in В. bifidum disintegrate (MB) after dilution and subsequent cultivation over the next 24 hours completely restored the ability to proliferate. The proliferative activity of the test cultures that were exposed to the extracts: filtrate of L. reuteri culture, grown in L. reuteri disintegrate (ML) and filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose (MLG), was significantly inhibited after dilution and subsequent cultivation. The inhibition indices calculated for the ML extract were: 25.9% (E. coli) and 53.0% (P. aeruginosa). Inhibition indices calculated for the MLG extract were: 62.0% (E. coli) and 96.9% (P. aeruginosa). MLG extract had more pronounced inhibitory effect on the proliferation of the test cultures than ML extract. All the studied extracts exerted significant inhibitory effect on the biofilm formation of the test cultures. Analysis of the results of the study shows that cell-free extracts of L. reuteri culture grown in its disintegrate without supplementation or supplemented with glycerol and glucose have the highest antimicrobial activity and can be used as metabiotics to prevent overgrowth of potentially pathogenic bacteria, as well as inoculation and proliferation of pathogenic gram-negative bacteria in the gastrointestinal tract. They can be used alone or in combination with cellular probiotics to enhance their probiotic action. This study encourages further careful investigation of the biochemical composition of cell-free extracts and clarifying the mechanism of their action.

Effects of long-term administration of Lactobacillus reuteri DSM-17938 on circulating levels of 5-HT and BDNF in adults with functional constipation

2019 ◽  
Vol 10 (2) ◽  
pp. 137-147 ◽  
Author(s):  
G. Riezzo ◽  
G. Chimienti ◽  
A. Orlando ◽  
B. D’Attoma ◽  
C. Clemente ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Functional Constipation ◽  
Probiotic Strain ◽  
Serum Levels ◽  
Double Blind ◽  
Term Administration ◽  
Probiotic Strains ◽  
S Allele ◽  
Item Scores

Accumulated evidence shows that some probiotic strains ameliorate functional constipation (FC) via the modulation of specific gastrointestinal peptide pathways. The aims of this study were to investigate: (1) the effects of long-term administration of Lactobacillus reuteri (LR) DSM 17938 on the serum levels of serotonin (5-HT) and brain-derived neurotrophic factor (BDNF); (2) the possible link between 5-HT, BDNF, and specific constipation-related symptoms; (3) whether genetic variability at the 5-HTT gene-linked polymorphic region (5-HTTLPR) and BDNF Val66Met loci could be associated with serum 5-HT and BDNF variations. LR DSM 17938 was administered to 56 FC patients for 105 days in a randomised, double-blind manner. The fasting blood samples were collected during the randomisation visit (V1), at day 15 (induction period, V2), day 60 (intermediate evaluation, V3), and day 105 (V4) and the Constipaq questionnaire (the sum of Constipation Scoring System (CSS) and patient assessment constipation quality of life (PAC-QoL)) was administered. A group of healthy subjects was enrolled as controls (HC). At V1, the mean serum 5-HT level in the whole patient group was significantly higher (P=0.027) than in HC subjects, while serum BDNF did not. At the end of probiotic administration (V4), 5-HT and BDNF levels were significantly lower than the initial values (V1) (P=0.008 and P=0.015, respectively). 5-HT and BDNF serum concentration were significantly associated (r=0.355; P=0.007). Neither 5-HT nor BDNF serum levels correlated with the CSS item scores and with the PAC-QoL. Lastly, the regression analysis demonstrated that the presence of the S allele of the 5-HTTLPR accounted for the reduction in the 5-HT concentration at V4. In conclusion, the long-term administration of LR DSM 17938 demonstrated that such a probiotic strain could improve FC by affecting 5-HT and BDNF serum concentrations.

scholarly journals Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG

2007 ◽  
Vol 73 (21) ◽  
pp. 6768-6775 ◽  
Author(s):  
Sarah Lebeer ◽  
Tine L. A. Verhoeven ◽  
M�nica Perea V�lez ◽  
Jos Vanderleyden ◽  
Sigrid C. J. De Keersmaecker
Keyword(s):  
Biofilm Formation ◽  
Culture Medium ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Lipoteichoic Acid ◽  
Microtiter Plate ◽  
Phenotypic Analysis ◽  
Lactobacillus Rhamnosus Gg ◽  
Abiotic Surfaces

ABSTRACTLactobacillus rhamnosusGG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover,L. rhamnosusGG displays very good in vitro adherence to epithelial cells and mucus. Here, we report thatL. rhamnosusGG is able to form biofilms on abiotic surfaces, in contrast to other strains of theLactobacillus caseigroup tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation byL. rhamnosusGG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation byL. rhamnosusGG.

scholarly journals Analysis of biofilm formation by intestinal lactobacilli

2015 ◽  
Vol 61 (6) ◽  
pp. 437-446 ◽  
Author(s):  
Magdaléna Slížová ◽  
Radomíra Nemcová ◽  
Marián Mad’ar ◽  
Jana Hadryová ◽  
Soňa Gancarčíková ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Spatial Organization ◽  
Lactobacillus Reuteri ◽  
Carbon Sources ◽  
Tween 80 ◽  
Microtiter Plate ◽  
Culture Conditions ◽  
De Man ◽  
Mrs Medium

In this study, the biofilm-forming potential of intestinal Lactobacillus reuteri strains under different culture conditions was characterized by microtiter plate biofilm assays. Moreover, the spatial organization of exogenously applied L. reuteri L2/6 (a pig isolate) at specific locations in gastrointestinal tract of monoassociated mice was investigated by fluorescence in situ hybridization. We did not detect biofilm formation by tested strains in nutrient-rich de Man–Rogosa–Sharpe (MRS) medium. On the contrary, a highly positive biofilm formation was observed in medium with lower accessibility to the carbon sources and lack of salts. The results obtained confirmed the significant role of Tween 80 and the quantity and nature of the sugars in the growth medium in biofilm formation. The omission of Tween 80 in MRS medium favored the formation of biofilm. Abundant biofilm formation was detected in the presence of lactose, galactose, and glucose. However, a gradual increase in sugar concentration triggered a significant decrease in biofilm formation. In addition, conditions related to the gastrointestinal environment, such as low pH and the presence of bile and mucins, highly modulated biofilm production. This effect seems to be dependent on the specificity and properties of the medium used for cultivation. From the evidence provided by this study we conclude that the biofilm formation capacity of L. reuteri is strongly dependent on the environmental factors and culture medium used.

scholarly journals In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts

2020 ◽  
Vol 11 (1) ◽  
pp. 93-97 ◽  
Author(s):  
O. V. Knysh ◽  
M. S. Pogorila ◽  
Y. V. Voyda
Keyword(s):  
Immune Response ◽  
Mononuclear Cells ◽  
Lactobacillus Reuteri ◽  
Peritoneal Macrophages ◽  
Bifidobacterium Bifidum ◽  
Structural Components ◽  
Intact Cells ◽  
Ldh Activity ◽  
Peripheral Mononuclear Cells

Recent studies have shown that alterations of the immune response in the gastrointestinal mucosa are key components of the mechanism of the probiotic action of beneficial bacteria. Most of the beneficial effects of probiotics are due to the action of their structural components and metabolites. Macrophages are first-line defense cells of the immune system, which not only participate in the detection, phagocytosis and destruction of harmful microorganisms, but also determine the nature of the subsequent immune response by presenting antigens to T-cells and initiating inflammation by releasing cytokines. We researched the effect of two types of cell-free extracts (CFEs) containing probiotic derivatives (structural components and metabolites of bacteria) Bifidobacterium bifidum 1 (BbCFE) and Lactobacillus reuteri DSM 17938 (LrCFE) on the activity of mouse peritoneal macrophages and on the ability of peripheral human blood mononuclear cells to produce cytokines. CFEs were obtained by culturing probiotics in their own disintegrates and then removing cells and cell debris by centrifugation and filtration. Peritoneal macrophages were isolated from mice. Some of them were infected in vitro by Salmonella thyphimurium. Uninfected and infected macrophages were incubated in culture medium containing (30% vol) or not containing CFEs at 37 °С in a microaerobic atmosphere (5% СО2) for 18 hours. After incubation, peritoneal macrophages were lysed. The obtained suspensions were centrifuged and supernatants were carefully collected. Macrophages activity was assessed by the nitrites level, superoxide dismutase (SOD), lactate dehydrogenase (LDH) activity and antiinflammatory cytokines levels in supernatants using spectrophotometric method. Peripheral mononuclear cells were isolated from the blood of healthy volunteers. The ability of peripheral mononuclear blood cells to produce antiinflammatory cytokines was evaluated after cell stimulation with lipopolysaccharide (LPS) and incubation with or without CFEs. Cytokine levels in supernatants were determined using enzyme-linked immunosorbent assay (ELISA). After infection with S. thyphimurium in macrophages, nitrite levels increased 5.5-fold, SOD activity 4.8-fold, and LDH 2-fold. Both studied CFEs exerted a similar effect on the macrophages’ activity. Addition of BbCFE to the incubation medium of infected macrophages resulted in a 4-fold decrease in nitrite levels, and the addition of LrCFE was accompanied by a decrease in nitrite levels to levels in intact cells. Under the influence of both CFEs, the activity of SOD and LDH was significantly reduced and did not differ significantly from the activity of these enzymes in intact cells. BbCFE and LrCFE did not have a significant effect on nitrite levels, SOD and LDH activity in intact macrophages. Under the influence of BbCFE, there was a 2-fold decrease in the production of TNF, a 2-fold increase in IL10 production, and a 30% increase in IL6 production by mononuclear cells. LrCFE caused a decrease in TNF production by 26.7% and IL6 by 36%, and IL10 by 1.9 times. Thus, the studied CFEs normalized the nitrite levels in peritoneal macrophages infected with S. thyphymurium and infection-induced activation of SOD and LDH enzymes. This demonstrates their ability to modulate oxidative processes in macrophages. In addition, under the influence of the investigated CFEs, there was a decrease in the production of pro-inflammatory cytokines (TNFα and IL-6) and increased production of anti-inflammatory cytokine (IL-10) by human peripheral mononuclear cells. The results of the study indicate the ability of CFEs by influencing the functions of innate immunity cells to restrict the inflammatory response and oxidative stress. Based on this, CFEs can be considered as promising agents for the treatment of inflammatory diseases.

Inhibitory effect of probiotic yeast Saccharomyces cerevisiae on biofilm formation and expression of α-hemolysin and enterotoxin A genes of Staphylococcus aureus

2019 ◽  
Author(s):  
Navid Saidi ◽  
Parviz Owlia ◽  
Seyed Mahmoud Amin Marashi ◽  
Horieh Saderi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Food Poisoning ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Microtiter Plate Assay ◽  
Probiotic Yeast ◽  
Mrsa Strains

Background and Objectives: Staphylococcus aureus, as an opportunistic pathogen, is the cause of a variety of diseases from mild skin infections to severe invasive infections and food poisoning. Increasing antibiotic resistance in S. aureus isolates has become a major threat to public health. The use of compounds produced by probiotics can be a solution to this problem. Thus, the purpose of this study was to investigate the effect of Saccharomyces cerevisiae on some virulence factors (biofilm, α-hemolysin, and enterotoxin A) of S. aureus. Materials and Methods: Supernatant and lysate extracts were prepared from S. cerevisiae S3 culture. Sub-MIC concen- trations of both extracts were separately applied to S. aureus ATCC 29213 (methicillin-sensitive S. aureus; MSSA) and S. aureus ATCC 33591 (methicillin-resistant S. aureus; MRSA) strains. Biofilm formation of these strains was measured by microtiter plate assay and expression level of α-hemolysin and enterotoxin A genes (hla and sea, respectively) using real-time PCR technique. Results: The supernatant extract has reduced both biofilm formation and expression of sea and hla genes, while lysate ex- tract had only anti-biofilm effects. The MRSA strain showed more susceptibility to yeast extracts than MSSA strain in all tests. Conclusion: The present study exhibited favorable antagonistic effects of S. cerevisiae S3, as a probiotic yeast, on MSSA and MRSA strains. Based on the findings of this study, the compounds produced by this yeast can be used to control S. aureus infections; however, further similar studies should be conducted to confirm the findings of the present study.

scholarly journals Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Fateme Davarzani ◽  
Zahra Yousefpour ◽  
Navid Saidi ◽  
Parviz Owlia
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Clinical Isolates ◽  
Minimum Inhibitory Concentrations ◽  
Alginate Production ◽  
Broth Microdilution Method ◽  
Encoding Genes

Background and Objectives: Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial viru- lence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expres- sion of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa. Materials and Methods: The broth microdilution method was used to determine the MIC of gentamicin for three P. aeru- ginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC con- centrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU). Results: Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expres- sion of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin. Conclusion: This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.

Enzymatic Inactivation of Pathogenic and Nonpathogenic Bacteria in Biofilms in Combination with Chlorine

2019 ◽  
Vol 82 (4) ◽  
pp. 605-614 ◽  
Author(s):  
MIN-JEONG KIM ◽  
EUN SEOB LIM ◽  
JOO-SUNG KIM
Keyword(s):  
Salmonella Typhimurium ◽  
Foodborne Pathogens ◽  
Biofilm Formation ◽  
Combined Treatment ◽  
Membrane Integrity ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Proteinase K ◽  
Food Contact Surfaces ◽  
Enzymatic Inactivation

ABSTRACT This study investigated the effects of enzyme application on biofilms of bacterial isolates from a cafeteria kitchen and foodborne pathogens and the susceptibility of Salmonella biofilms to proteinase K combined with chlorine treatment. For four isolates from a cafeteria kitchen (Acinetobacter, Enterobacter, and Kocuria) and six strains of foodborne pathogens (Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus), the inhibitory effect of enzymes on biofilm formation at 25°C for 24 h or the degradative efficacy of enzymes on 24-h mature biofilm at 37°C for 1 h in tryptic soy broth (TSB) was examined in a polystyrene microtiter plate. The effect of enzymes was also evaluated on a subset of these strains in 20 times diluted TSB (1/20 TSB) at 25°C. The working concentrations of five enzymes were 1 U/100 μL for α-amylase, amyloglucosidase, cellulase, and DNase and 1 milli-Anson unit/100 μL for proteinase K. In addition, 24-h mature Salmonella Typhimurium biofilm on a stainless steel coupon was treated with proteinase K for 1 h at 25°C followed by 20 ppm of chlorine for 1 min at 25°C. The results showed that certain enzymes inhibited biofilm formation by the kitchen-originated bacteria; however, the enzymatic effect was diminished on the mature biofilms. Biofilm formation of V. parahaemolyticus was suppressed by all tested enzymes, whereas the mature biofilm was degraded by α-amylase, DNase I, and proteinase K. Proteinase K was effective in controlling Salmonella biofilms, whereas a strain-dependent variation was observed in S. aureus biofilms. In 1/20 TSB, Enterobacter cancerogenus and Kocuria varians were more susceptible to certain enzymes during biofilm formation than those in TSB, whereas the enzymatic effect was much decreased on 24-h mature biofilms, regardless of nutrient conditions. Furthermore, synergistic inactivation of Salmonella Typhimurium in biofilms was observed in the combined treatment of proteinase K followed by chlorine. Live/Dead assays also revealed a decrease in density and loss of membrane integrity in Salmonella Typhimurium biofilms exposed to the combined treatment. Therefore, certain enzymes can control biofilms of isolates residing in a cafeteria kitchen and foodborne pathogens. This study demonstrates the potential of enzymes for the sanitation of food processing environments and of proteinase K combined with chlorine to control Salmonella biofilms on food contact surfaces.

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