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Taxonomic identification of microorganisms by capture and intrinsic fluorescence detection.

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading edge technologies described in this and the accompanying article. Microbe capture chips, used with a prototype fluorescence detector, are capable of statistically sampling the environment for pathogens (including spores), identifying the specific pathogens/exotoxins, and determining cell viability where appropriate.

Adhesins, Bacterial↗

Circulating endothelial cells in vascular disorders: new insights into an old concept.

The endothelial contribution to vascular disorders has been widely documented in experimental models. However, its implication in human pathology is difficult to investigate, owing to the paucity of noninvasive methods and of specific endothelial markers. The enumeration of circulating endothelial cells (CEC) released in peripheral blood after vascular injury represents a direct exploration of the endothelium. For this purpose, we have produced a monoclonal antibody (S-Endo 1), which recognizes CD 146, a molecule expressed on all types of human endothelial cells but absent from haemopoietic cells. Using this antibody, we have designed a specific and sensitive immunocapture test, which allowed us to detect high numbers of CEC in thrombotic, infectious or immunological disorders, while CEC were found to be very rare ( < 3/ml) in normal subjects. This quantitative approach using CEC might prove useful as a marker of vascular wall injury. Their enumeration is of interest in the clinical follow-up of vascular disorders, in the evaluation of therapeutic effectiveness or in the direct diagnosis of infectious diseases involving intra-endothelial microbial agents. Furthermore, an immunological and/or functional study of CEC could allow one to assess their procoagulant and proadhesive properties, as well as their viability, opening new perspectives for CEC investigation in vascular pathology.

Antigens, CD↗

Comparative responses of Pseudomonas stutzeri and Pseudomonas aeruginosa to antibacterial agents.

The sensitivity of six strains of Pseudomonas stutzeri (NCIMB 568, 10783, 11358, 11359, JM 302, JM 375) to cationic antiseptics, mercury compounds, the parabens, phenolics, EDTA and various antibiotics was compared with Pseudomonas aeruginosa NCIMB 8626. All Ps. stutzeri strains were highly sensitive to chlorhexidine diacetate, organomercurials and triclosan, but rather less so to quarternary ammonium compounds (QACs). They were also sensitive to other biocidal agents and more sensitive to many antibiotics than the strain of Ps. aeruginosa. There was little correlation between uptake of chlorhexidine diacetate or cetylpyridinium chloride by dense suspensions of organisms, leakage of intracellular constituents and loss of cell viability.

Anti-Bacterial Agents↗

Determination of molecular hydrogen in investigations of the susceptibility of Enterobacteriaceae to ampicillin.

One hundred and five enterobacterial isolates from urinary tract infections were examined for ampicillin-susceptibility by measurement of molecular hydrogen production with a palladium metal oxide semi-conductor (Pd-MOS sensor). Eighty-one per cent of hydrogen producing strains could be placed in the same susceptibility groups as recorded by disc-diffusion tests. However, it was harder to separate susceptible from intermediate types than to determine resistance. In all but five strains the hydrogen production allowed determination of viability within 5 h.

Ampicillin↗

Genetic analysis of Treponema denticola ATCC 35405 biofilm formation.

Treponema denticola is a major aetiological organism implicated in periodontal disease. The interaction of T. denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease. The interaction between T. denticola and P. gingivalis has been examined using a panel of T. denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model. T. denticola ATCC 35405 did not form detectable biofilms on various inert surfaces. However, the spirochaete was demonstrated to form a biofilm with preattached P. gingivalis 381. T. denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P. gingivalis. A T. denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T. denticola mutant which does not possess the major outer sheath protein (Msp). The T. denticola lrrA mutant was only moderately defective in forming mixed biofilms with P. gingivalis. However, the T. denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation. In contrast, T. denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Bacterial Proteins↗

Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid.

The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined. Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain. Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid. However, the uvrB gene also plays a role in maintaining cell viability.

DNA Repair↗

[Combined activity of meliacin and foscarnet against different strains of herpes simplex virus type 1 using a three-dimensional model].

We evaluated the in vitro antiviral activity of meliacin combined with foscarnet on the herpes simplex type 1 (HSV-1) strains F and B2006 (tk-) replication. The effective concentrations for 50% inhibition of HSV-1 (F) were 12.5 micrograms/ml for meliacin and 15.7 micrograms/ml for foscarnet, while for HSV-1 (B2006) were 3.1 micrograms/ml and 126 micrograms/ml, respectively. The data were analyzed for quantitation of synergism, additivity, and antagonism of multiple drug effect by the three-dimensional model. Some of the meliacin -foscarnet combinations synergistically inhibited HSV-1 (F) and HSV-1 (B2006) replication in vitro at concentrations that did not reduce cellular viability.

Animals↗

[An ecologic hygiene assessment of the microbiological processes in soil contaminated with sulfonol and lead].

Alteration of the microbial coenosis and biological activity of different types of soils as well as peculiarities of survival of sanitary-significant and pathogenic microorganisms under the effect of sulphonol and a sulphonol-lead complex have been studied in the laboratory model experiment. The most sensitive tests were as follows: the number of nitrifying bacteria; nitrifying, proteolytic, dehydrogenase and cellulase activity; survival of salmonella and lactose-positive colibacilli. Under the effect of the tested sulphonol (0.5 and 3 mg/l) and lead (0.1 and 2 mg/l) concentrations the self-purification processes in soil were not substantially disturbed, though surfactants negatively affected viability of enterobacteria.

Benzenesulfonates↗

Efficiency of pulsed UV light for microbial decontamination of food powders.

The aim of this study was to evaluate the efficiency of pulsed light on the destruction of dried microorganisms on fluidized glass beads and to determine treatment parameters (energy level, water activity, final product quality) for process optimization. The applied drying method allowed microorganisms to remain viable on glass beads or dried powdered products with viability yields approaching 100%. The pulsed UV light system enabled an efficient fluidization of food powders, even for granular products (up to 5 mm diameter) and avoided shadowed areas. For Saccharomyces cerevisiae decontamination, the dose effect of UV rays was preponderant with glass beads and quartz plate, and in this case, 58 J/cm2 were required to decrease the microbial population by 7 log. For colored food powders (black pepper and wheat flour), the thermal effect of pulsed light dominated the UV effect.

Consumer Product Safety↗

Compared tolerance to osmotic stress in various microorganisms: towards a survival prediction test.

The osmotic tolerance of microbial cells of different microorganisms was investigated as a function of glycerol concentration and temperatures. Cells displayed specific sensitivity to dehydration in glycerol solutions. The viability of Gram-negative strains (Escherichia coli, Bradyrhizobium japonicum), Gram-positive strains (Lactobacillus plantarum, L. bulgaricus), and yeasts (Saccharomyces cerevisiae, Candida utilis) decreased with increasing osmotic pressure. For each strain, a characteristic osmotic pressure threshold causing a loss of 40% of the population at the growth temperature was determined: 26-40 MPa for E. coli, 15-25 MPa for B. japonicum, 7-15 MPa for L. bulgaricus, 40-133 MPa for L. plantarum, 50-100 MPa for S. cerevisiae, and 15-26 MPa for C. utilis. Because this threshold varies with temperature, it was possible to construct a diagram that could be helpful to the determination of the sensitivity of each strain to osmotic stress as a function of osmotic pressure and temperature.

Gram-Negative Bacteria↗

Calorimetric determination of inactivation parameters of micro-organisms.

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.

Calorimetry, Differential Scanning↗

Physical simulation for low-energy astrobiology environmental scenarios.

Speculations about the extent of life of independent origin and the potential for sustaining Earth-based life in subsurface environments on both Europa and Mars are of current and relevant interest. Theoretical modeling based on chemical energetics has demonstrated potential options for viable biochemical metabolism (metabolic pathways) in these types of environments. Also, similar environments on Earth show microbial activity. However, actual physical simulation testing of specific environments is required to confidently determine the interplay of various physical and chemical parameters on the viability of relevant metabolic pathways. This testing is required to determine the potential to sustain life in these environments on a specific scenario by scenario basis. This study examines the justification, design, and fabrication of, as well as the culture selection and screening for, a psychrophilic/halophilic/anaerobic digester. This digester is specifically designed to conform to physical testing needs of research relating to potential extent physical environments on Europa and other planetary bodies in the Solar System. The study is a long-term effort and is currently in an early phase, with only screening-level data at this time. Full study results will likely take an additional 2 years. However, researchers in electromagnetic biosignature and in situ instrument development should be aware of the study at this time, as they are invited to participate in planning for future applications of the digester facility.

Acetates↗

Microbial contamination of water-soaked cotton gauze and its cause.

Seven in-use cotton gauze samples and three cotton balls soaked in sterile distilled water in canisters were investigated 7 days after they were prepared in hospital. All samples were contaminated with bacteria including 10(6) to 10(7) colony forming units/ml of Pseudomonas aeruginosa. In vitro viability tests using cotton gauze and cotton balls soaked in sterile distilled water revealed rapid proliferation of P. aeruginosa, Serratia marcescens and Candida albicans. Since the cotton gauze and the cotton balls were soaked in water containing nutrients, such as protein and glucose, these materials may be readily contaminated with bacteria including P. aeruginosa. Thus, when using cotton gauze and cotton balls containing water, microbial contamination should be expected.

Candida albicans↗

Behavior of Listeria monocytogenes in pH-modified chicken salad during refrigerated storage.

The growth and inactivation kinetics of L. monocytogenes were evaluated at pH 4.0, 4.6, and 5.2 during storage at 5.0 degrees C, 7.2 degrees C, and 21.1 degrees C (41 degrees F, 45 degrees F, and 70 degrees F). Using commercially produced pasteurized chicken salad, the authors adjusted the pH levels with acetic acid or sodium acetate. Samples of 25 g each of the pH-modified salad were inoculated to approximately 1 x 10(6) cells per gram with a three-strain mixture of L. monocytogenes and stored for up to 119 days. Samples were enumerated for L. monocytogenes according to the Food and Drug Administration-modified most-probable-number (MPN) procedure, and log MPN was plotted against time. Inactivation was seen at all pH levels and at all temperatures. At 21.1 degrees C, a 6-log reduction was seen after 14 days at pH 4.0, after 52 days at pH 4.6, and after 38 days at pH 5.2. Inactivation at 21.1 degrees C began within hours or days at pH 4.0, and after a lag phase of 10 to 12 days at pH 4.6 and 5.2. Inactivation was slower in cold-storage temperatures. At 7.2 degrees C, a microbial reduction of 1.1 log (pH 5.2) and > 3 log (pH 4.0 and 4.6) was observed at 119 days. At 5 degrees C, a 7.5-log reduction was observed at 24 days at pH 4.0. At pH levels of 4.6 and 5.2, however, only a 4-log reduction was found at 119 days. The data generated in this study may be used to develop predictive models that could specifically address the interactions of pH and storage temperature on the viability of L. monocytogenes in prepared salads.

Acetic Acid↗

Cryovial with partial membrane sealing can prevent liquid nitrogen penetration in submerged storage.

Cryopreservation is one of the fundamental techniques in life science. To preserve the viability of cells and tissues, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the non-sterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. To prevent these drawbacks while retaining the benefit of constant ultra-low temperature in submerged liquid nitrogen, we used a heat-sealable membrane to cover the upper portion of vials. After heat-sealing, the vials were completely free of liquid nitrogen penetration in the submerging test. Moreover, the sealing process did not affect the cell viability. This modified protocol provides an easy and efficient tool to ensure the integrity of biospecimens in long-term storage without interfering with existing cryobox storage systems.

Animals↗