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PubMedDate not supplied

Detection of extracellular proteases from microorganisms on agar plates.

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

Agar↗
PubMedDate not supplied

Susceptibility of some oral microorganisms to chlorhexidine and paramonochlorophenol.

Since the use of antimicrobial agents is required in endodontic therapies, this study aimed at determining the minimum inhibitory concentrations (MICs) of chlorhexidine digluconate and paramonochlorophenol (PMC) against microorganisms commonly found in endodontic infections. Both agents were tested by agar dilution tests against Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella denticola and Prevotella melaninogenica. The MIC of chlorhexidine ranged from 2.67 to 80.00 microg/ml, and the MIC of PMC from 46.67 to 213.33 microg/ml. The highest MIC value of PMC was detected for E. faecalis whereas E. coli was the most susceptible microorganism to this agent. The highest MIC values of chlorhexidine were observed for P. aeruginosa whereas E. coli and P. denticola were the most susceptible microorganisms to this agent. Since the MIC values observed are much lower than the concentrations currently used in the endodontic therapy, it is suggested that both agents are effective in reducing the microbiota in the root canal.

Anti-Infective Agents, Local↗
PubMedDate not supplied

Effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust.

High levels of dust and microorganisms are known to be associated with animal confinement rearing facilities. Many of the microorganisms are carried by dust particles, thus providing an excellent vector for horizontal disease transmission between birds. Two environmentally controlled rooms containing female broiler breeder pullets (n = 300) were used to evaluate the effectiveness of an electrostatic space charge system (ESCS) in reducing airborne dust and gram-negative bacteria levels over an 8-wk period (starting when the birds were 10 wk old). The ESCS was used to evaluate the effectiveness of reducing airborne microorganism levels by charging airborne dust particles and causing the particles to be attracted to grounded surfaces (i.e., walls, floor, equipment). The use of the ESCS resulted in a 64% mean reduction in gram-negative bacteria. Airborne dust levels were reduced an average of 37% over a 1-wk period in the experimental room compared with the control room on the basis of samples taken every 10 min. The reductions of airborne dust and bacteria in this study are comparable with earlier results obtained with the ESCS in commercial hatching cabinets and experimental caged layer rooms, suggesting the system could also be applied to other types of enclosed animal housing.

Air Microbiology↗
PubMed2003

Combination of microautoradiography and fluorescence in situ hybridization for identification of microorganisms degrading xenobiotic contaminants.

One of the central topics in environmental bioremediation research is to identify microorganisms that are capable of degrading the contaminants of interest. Here we report application of combined microautoradiography (MAR) and fluorescence in situ hybridization (FISH). The method has previously been used in a number of systems; however, here we demonstrate its feasibility in studying the degradation of xenobiotic compounds. With a model system (coculture of Pseudomonas putida B2 and Sphingomonas stygia incubated with [14C] o-nitrophenol), combination of MAR and FISH was shown to be able to successfully identify the microorganisms degrading o-nitrophenol. Compared with the conventional techniques, MAR-FISH allows fast and accurate identification of the microorganisms involved in environmental contaminant degradation.

Autoradiography↗
PubMed1998

Periodontal status and serum antibody responses to oral microorganisms in Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease characterized by keratoconjunctivitis sicca and xerostomia. Rapid bacterial plaque accumulation occurs in Sjögren's syndrome patients due to decreases in salivary flow rate. The purpose of this study was to evaluate the periodontal status of patients with Sjögren's syndrome and evaluate serum antibody responses to selected oral microorganisms, including major periodontopathogens, compared to healthy controls. Seventeen Sjögren's syndrome patients and 14 healthy subjects were included in the study. Plaque (PL), sulcular bleeding (SBI), periodontal index scores (PI), probing depths (PD), and total number of teeth were recorded. An ELISA was used to determine the serum IgG antibody level to a panel of 13 oral microorganisms. Significantly higher PL, SBI, PD, and PI scores, as well as an increased number of lost teeth were observed in patients with Sjögren's syndrome compared to healthy subjects (P <0.0001). Antibody levels to Streptococcus oralis were significantly lower in Sjögren's syndrome patients than controls (P <0.0002). These patients exhibited significantly elevated antibody levels to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis compared to controls (P <0.006 to 0.0004). Our findings indicate that Sjögren's syndrome patients have established periodontal disease and serum antibody responses to oral microorganisms previously identified as periodontopathogens in systemically healthy subjects. These results suggest that Sjögren's syndrome may affect bacterial colonization in plaque and contribute to increased periodontal disease in this compromised population.

Adolescent↗
PubMed1999

Periodontal findings and systemic antibody responses to oral microorganisms in Behçet's disease.

BACKGROUND: Behçet's disease is a multisystem disorder of unknown etiology, affecting predominantly the oral mucosa, skin, and eyes. Recurrent and painful episodes of oral ulcerations interfere with regular oral hygiene leading to rapid bacterial plaque accumulation. The aims of this study were to evaluate the periodontal status of patients with Behçet's disease and determine serum antibody responses to selected oral microorganisms, including major periodontopathogens in these patients. METHODS: Thirty-three patients with Behçet's disease and 15 healthy subjects were included in the study. Plaque, sulcular bleeding, periodontal index scores, probing depths, and total number of teeth were recorded. Serum IgG antibody levels to a panel of 13 oral microorganisms were determined. RESULTS: Significantly higher values for each of the clinical measures were observed in patients with Behçet's disease compared to healthy subjects (P <0.0001). Antibody levels to selected members of plaque, including Actinomyces viscosus, Streptococcus mutans, Streptococcus sanguis, Streptococcus oralis, Eikenella corrodens, Campylobacter rectus, and Prevotella intermedia were significantly lower in patients with Behçet's disease than in controls (P <0.001-0.05). In contrast, these patients exhibited significantly elevated antibody levels to Actinobacillus actinomycetemcomitans Y4 compared to controls (P <0.01). CONCLUSIONS: Our data indicate that the patients with Behçet's disease generally exhibit clinical findings of established periodontal disease. Decreased antibody responses to early colonizers of both supra- and subgingival plaque were observed along with the elevation in antibody levels to A. actinomycetemcomitans. These results suggest that the bacterial plaque ecology and/or immune responses to these microorganisms may be affected in Behçet's disease which could lead to changes in the expression of periodontal disease.

Actinomyces viscosus↗
PubMed2004

Biosorption and recycling of gold using various microorganisms.

In order to obtain basic information on the biosorption and recycling of gold from aqueous systems using microbial cells, the biosorption of gold by various microorganisms was investigated. Of 75 strains of microorganisms tested (25 bacteria, 19 actinomycetes, 17 fungi and 14 yeasts), high abilities of gold biosorption from a solution containing hydrogen tetrachloroaurate (III) were found in some gram-negative bacterial strains, such as Acinetobacter calcoaceticus, Erwinia herbicola, Pseudomonas aeruginosa, and P. maltophilia. Most of the gram-positive bacteria, actinomycetes, fungi and yeasts had a lower ability for gold biosorption than gram-negative bacteria. On the other hand, all of the microorganisms tested adsorbed far smaller amounts of gold from a solution containing gold dicyanoaurate (I). The biosorption of gold from a solution containing hydrogen tetrachloroaurate (III) using P. maltophilia having a high adsorbing ability for gold was very rapid and was affected by the pH of the solution, external gold concentration, and cell amounts. P. maltophilia cells immobilized with polyacrylamide gel also have a high ability for gold biosorption. The gold adsorbed on the immobilized cells is easily desorbed with 0.1 M thiourea solution. The immobilized P. maltophilia cells can be used repeatedly in biosorption-desorption cycles.

Adsorption↗
PubMed1995

Effect of malate on in vitro mixed ruminal microorganism fermentation.

The objective of this study was to evaluate the effects of different concentrations of DL-malate (disodium salt) on the in vitro mixed ruminal microorganism fermentation of soluble starch or cracked corn. Ruminal fluid was collected from a steer fed 6.8 kg of forage and 2.3 kg of concentrate supplement once daily, and mixed ruminal microorganisms were incubated in anaerobic media (40 mL) that contained 20% (vol/vol) ruminal fluid in batch culture for 24 h at 39 degrees C. Malate was added to the incubation bottles (n = 4) to achieve final concentrations of 0, 4, 8, and 12 mM. When mixed ruminal microorganisms were incubated with only DL-malate as the substrate, final pH numerically increased, propionate and total VFA concentrations increased (P < .05), and the acetate:propionate ratio decreased (P < .05) as the concentration of DL-malate increased from 0 to 12 mM. Fermentation of cracked corn in the presence of 8 or 12 mM DL-malate resulted in an increase (P < .05) in final pH and propionate concentration. Total VFA tended to increase (P < .21), whereas final concentrations of L-lactate numerically decreased. In the case of soluble starch, 8 and 12 mM DL-malate caused a decrease (P < .05) in methane concentration. When only ruminal fluid (no added anaerobic medium) was used as the inoculum rather than 20% ruminal fluid medium, similar results for final pH, propionate, L-lactate, and total VFA were observed for soluble starch and corn incubations treated with DL-malate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Effects of organic acid and monensin treatment on in vitro mixed ruminal microorganism fermentation of cracked corn.

The objective of this study was to determine the effects of organic acids and monensin on the in vitro fermentation of cracked corn by mixed ruminal microorganisms. Ruminal fluid was collected from a steer fed 36.3 kg of wheat silage and 4.5 kg of concentrate supplement once daily. Mixed ruminal microorganisms were incubated in anaerobic media that contained 20% (vol/vol) ruminal fluid and .4 g of cracked corn. Incubations were carried out in batch culture for 24 h at 39 degrees C. Organic acids (L-aspartate, fumarate, and DL-malate) were added to serum bottles (n = 4) to achieve final concentrations of 0, 4, 8, or 12 mM. Monensin, dissolved in ethanol, was included in serum bottles at a final concentration of 0 or 5 ppm of culture fluid. The addition of 8 and 12 mM organic acids to cracked corn fermentations increased final pH (P < .05), tended to increase total gas production and CO2 concentration, and decreased the acetate:propionate ratio (P < .05). Organic acids tended to decrease methane concentrations and hydrogen concentration was not altered. DL-Malate addition at all levels reduced (P < .05) lactate accumulation. Additive effects of monensin and organic acids were observed in some fermentations. In conclusion, organic acid addition to in vitro mixed ruminal microorganism fermentations yielded beneficial results independent of monensin treatment by decreasing the acetate: propionate ratio and increasing final pH.

Acetates↗
PubMed1999

Effects of laidlomycin propionate and monensin on the in vitro mixed ruminal microorganism fermentation.

The objective of this study was to compare the effects of laidlomycin propionate and monensin on the in vitro fermentation of ground corn, Trypticase, or alfalfa hay by mixed ruminal microorganisms. Ruminal fluid was collected from two steers fed 9.27 kg DM of a high-concentrate (62.2% ground corn and 17.4% cottonseed hulls) diet per day and composited. In the first study, no ionophore was included in the diet; the diet in the second study contained 11.1 g of laidlomycin propionate per ton of feed. The animals were allowed an adjustment period of 14 d for each dietary treatment before samples were collected. When ruminal fluid from unadapted animals was used, both monensin and laidlomycin propionate decreased (P<.05) CH4 concentration and the acetate:propionate ratio with ground corn and alfalfa hay. Monensin reduced (P<.05) in vitro dry matter disappearance of alfalfa and increased (P<.05) final pH in the ground corn and alfalfa hay fermentations. Both laidlomycin propionate and monensin decreased (P<.05) concentrations of acetate, propionate, isobutyrate, isovalerate, CH4, and NH3 in Trypticase fermentations. When ruminal fluid from adapted animals was used, both ionophores still reduced the concentrations of most fermentation products. However, there was generally less inhibition compared with fermentations inoculated with unadapted mixed ruminal microorganisms. In the presence of 5 mM maltose, mixed ruminal bacteria produced high concentrations (10 to 11 mM) of lactate, and addition of both ionophores to these fermentations was effective in reducing (P<.05) lactate production. In conclusion, laidlomycin propionate alters the mixed ruminal microorganism fermentation in a manner similar to monensin, but, at the concentrations used in this study, monensin seemed to be a more potent inhibitor.

Animal Feed↗
PubMed1989

Potentially pathogenic microorganisms in the oral cavity during febrile episodes in immunocompromised patients with haematologic malignancies.

The purpose of the study was to describe the occurrence of potentially pathogenic microorganisms during febrile episodes in haematologic patients, who had been treated with anti-neoplastic drugs within the previous 4 weeks. During a 9-month period 78 consecutive febrile episodes occurred in 46 patients who were examined daily. On day 1, before antibiotic treatment was started, members of the normal flora were found in 83%, Candida spp. in 72%, Enterobacteriaceae in 29%, Pseudomonas aeruginosa in 8%, Staphylococcus aureus in 14%, coagulase-negative staphylococci in 26% and enterococci in 17% of the febrile episodes. During the subsequent antibiotic treatment, the prevalence of members of the normal oral flora was markedly reduced from day 1 (83%) to day 7 (6%), and the normal flora was replaced by potentially pathogenic microorganisms. A rise was found in the prevalences of Candida spp. (from 72 to 92%) and coagulase-negative staphylococci (from 26 to 49%) from day 1 to day 4. The prevalences of Enterobacteriaceae and P. aeruginosa showed no day-to-day changes, and were not different in patients hospitalized for more or less than 24 h prior to inclusion into the study, suggesting that their detection was not due to acquisition during hospitalization or antibiotic treatment. The oral occurrence of species of microorganisms regularly found in septicaemia, suggests that the oral cavity may play a role as a port of entry of septicaemia in patients with haematologic malignancies.

Adult↗
PubMed1998

Functional changes in THP-1 human monocytic cells after stimulation with lipopolysaccharide of oral microorganisms and granulocyte macrophage colony stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using F1TC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10-11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2-3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nucleatum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.

Cell Line↗
PubMed1981

Gas chromatography in the identification of microorganisms and diagnosis of infectious diseases.

Gas-liquid chromatography (GLC) continues to find increasing applications in the characterization of microorganisms and the diseases associated with them. GLC has been used to characterize microorganisms through qualitative and quantitative analyses of cellular structural components and soluble extracts and metabolic by-products. Chromatographic patterns, or fingerprints, can be used to differentiate between very closely related microorganisms, even strain differences, and provides a potentially powerful tool for future taxonomic studies and more precision in definitive microbial classification. However, the most valuable use of GLC is in the identification of disease through patterns obtained by direct analysis of body fluids. Chromatographic fingerprints of microbial metabolites and compounds associated with the host response to infection and even noninfectious disease can be used to develop relatively simple GLC diagnostic methods. These methods are specific, sensitive, and rapid. This review examines the use of GLC for identification of infectious diseases through the analysis of body fluids, spent culture media, and cellular materials and suggest other clinical areas where its diagnostic potential has yet to be developed.

Bacterial Infections↗
PubMed2000

Influence of fluorescence of bacteria stained with acridine orange on the enumeration of microorganisms in raw milk.

The staining of gram-positive and gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method. The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.

Acridine Orange↗
PubMed1989

Effect of threonine and glycine concentrations on threonine aldolase activity of yogurt microorganisms during growth in a modified milk prepared by ultrafiltration.

To evaluate the combined effects of threonine and glycine concentrations during growth on threonine aldolase activity (EC 2.1.2.1) of yogurt microorganisms, Streptococcus thermophilus and Lactobacillus bulgaricus, a modified milk growth medium was prepared using UF to deplete the free amino acid level. Threonine and glycine were added according to a 2x2x2 factorial design at 5 or 195 microg.ml(-1) along with a standard amino acid mixture. Acetaldehyde production and threonine aldolase activity were evaluated utilizing headspace gas chromatography. Results showed that threonine and glycine concentrations did not affect growth or titratable acidity. The high concentration of threonine in combination with low glycine in the growth medium resulted in increased acetaldehyde synthesis by both microorganisms. Conversely, high glycine with low threonine decreased acetaldehyde synthesis. High threonine and low glycine increased threonine aldolase activity of cell-free extracts from S. thermophilus and L. bulgaricus, whereas high glycine and low threonine reduced threonine aldolase activity of both microorganisms.

Acetaldehyde↗
PubMed1999

Effects of a Saccharomyces cerevisiae culture on in vitro mixed ruminal microorganism fermentation.

Previous research has shown that Saccharomyces cerevisiae culture increases lactate utilization and cellulose digestion by pure cultures of ruminal bacteria. Based on these pure culture results, in vitro mixed ruminal microorganism fermentations were conducted to determine the effects of 0.35 and 0.73 g/L of Sacc. cerevisiae culture on the fermentation of ground corn, maltose, alfalfa hay, bermudagrass hay, and lactate. In addition, experiments were performed to evaluate the effects of Sacc. cerevisiae culture and monensin on the mixed ruminal microorganism fermentation. In the presence of ground corn, both concentrations of Sacc. cerevisiae culture had little effect on final pH or fermentation products, except the 0.35 g/L treatment increased valerate concentration. Saccharomyces cerevisiae culture had little effect on final pH or fermentation products in maltose or lactate fermentations. When alfalfa hay was the substrate, 0.73 g/L of Sacc. cerevisiae culture increased propionate concentration and both treatments decreased the acetate to propionate ratio. In the case of Coastal bermudagrass hay, 0.73 g/L Sacc. cerevisiae culture increased concentrations of acetate, propionate, CH4, butyrate, isovalerate, valerate, and decreased the acetate to propionate ratio, whereas both treatments increased total volatile fatty acid concentrations. Similar to alfalfa hay, in vitro dry matter disappearance of Coastal bermudagrass hay was numerically increased in the presence of Sacc. cerevisiae culture. Monensin altered the fermentation by decreasing concentrations of CH4 and lactate and increasing concentrations of propionate. There was no interaction between Sacc. cerevisiae culture and monensin. In conclusion, the incorporation of Sacc. cerevisiae culture into mixed ruminal microorganism fermentations of ground corn, maltose, or lactate had little effect on final pH and fermentation products. However, in the presence of alfalfa hay or Coastal bermudagrass hay Sacc. cerevisiae culture increased concentrations of several fermentation products and numerically increased in vitro dry matter disappearance of forage fiber.

Acetates↗
PubMed2001

Comparison between xanthine oxidases from buttermilk and microorganisms regarding their ability to generate reactive oxygen species.

Xanthine oxidase (XO) forms uric acid from xanthine. It is assumed that at the same time oxygen is reduced by the XO to reactive oxygen species (ROS), mainly to .O2- and to H2O2. Under certain conditions such ROS can be highly damaging to cellular structures. Therefore, XO was frequently used as a model system, in which the impact of ROS on cellular compounds and structures has been investigated. In this in vitro study xanthine oxidases from buttermilk and from microorganisms were compared regarding their ability to generate ROS. It could be shown that both enzymes are able to transform xanthine to uric acid but differ significantly in their reductive properties to oxygen. XO from buttermilk reduces oxygen to both .O2- and H2O2 whereas XO from microorganisms generates H2O2, but fails to form .O2-. Since .O2- are involved in maintaining transition metal-mediated formation of hydroxyl radicals (.OH) from H2O2, we conclude that XO from microorganisms is therefore largely unsuitable in studies investigating just the interaction of .O2- with other ROS on cellular compounds.

Animals↗
PubMed1990

In vitro adherence of microorganisms to denture base resin with different surface texture.

We examined the effects of various denture base resin surface textures on the adherence of microorganisms. S. sanguis and B. gingivalis adhered in greater amounts to the denture base resin than the other microorganisms tested. As to bacterial adherence according to polishing state, S. oralis, B. gingivalis C-101, and B. intermedius C-001 more adhered to the No. 400 paper-polished surface than to the buff-polished and smoothening-treated surfaces. S. sanguis less adhered to the smoothening-treated surface. S. mitis and C. albicans, on the other hand, more adhered to the smoothening-treated surface. For the other microorganisms tested, no relationship was observed between surface texture and bacterial adherence. The fall-off test revealed no remarkable differences in the fall-off of S. sanguis and B. gingivalis C-101 by the types of surface treatment. However, the fall-off of C. albicans was poorest from the No. 400 paper-polished surface. These results indicate that smoothening the denture base surface is important for denture plaque control.

Bacterial Adhesion↗