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PubMed1995

Activity and purification of linenscin OC2, an antibacterial substance produced by Brevibacterium linens OC2, an orange cheese coryneform bacterium.

An orange cheese coryneform bacterium isolated from the surface of Gruyère of Comté and identified as Brevibacterium linens produces an antimicrobial substance designated linenscin OC2. This compound inhibits gram-positive food-borne pathogens including Staphylococcus aureus and Listeria monocytogenes but is not active against gram-negative bacteria. Linenscin OC2 caused viability loss and lysis of the test organism, Listeria innocua. Electron microscopy showed that linenscin OC2 induces protoplast formation and cell lysis. The native substance is resistant to proteolytic enzymes, heat, and organic solvents and stable over a wide range of pH. The molecular weight of the native linenscin OC2 was estimated by gel chromatography to be over 285,000. Linenscin OC2 was purified by ammonium sulfate precipitation, 2-propanol extraction, and reverse-phase chromatography. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass under 2,412 Da. Molecular mass was determined to be 1,196.7 Da by mass spectrometry. Amino acid composition analysis indicated that linenscin OC2 may contain 12 residues.

Amino Acids↗

PubMed1990

Isolation of purified large mammal and human islets of Langerhans.

Previous studies have shown that the yield and viability of islets obtained from human or large mammal pancreas depends upon techniques used for islet isolation and upon factors that affect the quality of the donor pancreas. In the present studies, the efficacy of collagenase digestion by ductal perfusion or automated techniques was compared in a canine model of purified islet isolation. The ductal perfusion technique was then developed for human pancreas which was excised before (n = 8) or after (n = 8) multiple organ procurement and without in situ arterial perfusion of hypothermic preservation solution or significant cold storage. Studies with canine pancreas showed that ductal perfusion yielded 2.0 +/- 0.7 microL of islets/g of pancreas and when combined with automated digestion, yields improved to 3.6 +/- 0.8 microL/g (versus perfusion alone, not significant). The yield and viability of human islets was improved when the pancreas was excised before procurement of other donor organs. Results of islet isolation from eight consecutive human pancreases procured in this manner revealed a total yield of 355.2 +/- 44 x 10(3) islets, corresponding to 5345 +/- 600 islets/g (+/- SEM, mean islet diameter 150 microns). Six of eight Ficoll purification attempts succeeded, yielding 186.6 +/- 31 x 10(3) islets of purity ranging from 45-60%. Perifusion with glucose elicited biphasic insulin secretion with a three-fold rise. Islets from two of the isolations were utilized to initiate a clinical trial of islet transplantation in insulin-dependent diabetes mellitus.

Animals↗

PubMed2005

Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons.

Thymol is a naturally occurring phenolic monoterpene known for its anti-microbial and anti-oxidant properties. It is used in dental practice and in anaesthetic halothane preparations. Recent studies have reported enhanced GABA(A) receptor-operated chloride channel activity and increased binding affinity of [(3)H]flunitrazepam in the presence of thymol. In the present work, we more closely examined the pharmacological action of thymol on the native GABA(A) receptor by using primary cultures of cortical neurons. Thymol enhanced GABA-induced (5 microM) chloride influx at concentrations lower than those exhibiting direct activity in the absence of GABA (EC(50) = 12 microM and 135 microM, respectively). This direct effect was inhibited by competitive and non-competitive GABA(A) receptor antagonists. Thymol increased [(3)H]flunitrazepam binding (EC(50) = 131 microM) and showed a tendency to increase [(3)H]muscimol binding. These results confirm that thymol is a positive allosteric modulator of the GABA(A) receptor. The thymol structural analogues menthol and cymene, which lack an aromatic ring or a hydroxyl group, did not affect [(3)H]flunitrazepam binding. Using a pharmacophoric model that includes a hydrogen bond donor group as well as an aromatic ring with two aliphatic substituents, we propose to demonstrate the molecular essential features of these compounds to interact with GABA(A) receptors. Thymol (0-1 mM) did not affect cellular viability.

Anesthetics, Intravenous↗

PubMed2003

Viability of microencapsulated bifidobacteria in simulated gastric juice and bile solution.

Microencapsulated cells of Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 were prepared by spray drying the cell suspension containing the test organism and 10% (w/w) of the carrier material of either gelatin, soluble starch, skim milk or gum arabic. Survival of these microencapsulated and free cells of bifidobacteria in simulated gastric juice (pH 2.0 and 3.0) and bile solution (0.5% and 2.0%) was then examined. B. infantis CCRC 14633 was more susceptible than B. longum B6 to the simulated gastric environment and bile solution tested. Microencapsulated bifidobacteria exhibited a lower population reduction than free cells during exposure to simulated gastric environment and bile solution. This phenomenon was most pronounced when the test organism was exposed to gastric juice at pH 2.0 or 2.0% bile solution. Moreover, it was also observed that the protective effect exerted by encapsulation with spray drying varied with the carriers used and the strains of bifidobacteria.

Animals↗

PubMedDate not supplied

Susceptibility testing of Cryptococcus neoformans using the urea broth microdilution method.

An urea broth microdilution method to assay the susceptibility of Cryptococcus neoformans to antifungal drugs was newly developed. Using this method, urease activity of the fungus was measured instead of the viability by checking colony development. The urease activities were indicated by colour changes in optical density at 545 nm. The end point in this assay was considered as 99% inhibitory concentration. When we measured antifungal activities of the three drugs against 16 isolates of Cr. neoformans using this assay method, mean minimum-inhibitory concentrations (MICs) of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.008 microgram ml-1 and 0.25 microgram ml-1 respectively. This assay method resulted in higher sensitivity in MICs of the three antifungal drugs than the broth microdilution method recommended by the Committee for Laboratory Standards of the Japanese Society for Medical Mycology. The results obtained using this assay method support the more effective evaluation of antifungal substances in susceptibility testing of Cr. neoformans.

Antifungal Agents↗

PubMedDate not supplied

Immunological status of ENL (erythema nodosum leprosum) patients: its relationship to bacterial load and levels of circulating IL-2R.

Recent data suggest that the clinical course of reactional states in leprosy is closely related to the cytokine profile released locally or systemically by the patients. In the present study, patients with erythema nodosum leprosum (ENL) were grouped according to the intensity of their clinical symptoms. Clinical and immunological aspects of ENL and the impact of these parameters on bacterial load were assessed in conjunction with patients' in vitro immune response to mycobacterial antigens. In 10 out of the 17 patients tested, BI (bacterial index) was reduced by at least 1 log from leprosy diagnosis to the onset of their first reactional episode (ENL), as compared to an expected 0.3 log reduction in the unreactional group for the same MDT (multidrug therapy) period. However, no difference in the rate of BI reduction was noted at the end of MDT among ENL and unreactional lepromatous patients. Accordingly, although TNF-alpha (tumor necrosis factor) levels were enhanced in the sera of 70.6% of the ENL patients tested, no relationship was noted between circulating TNF-alpha levels and the decrease in BI detected at the onset of the reactional episode. Evaluation of bacterial viability of M. leprae isolated from the reactional lesions showed no growth in the mouse footpads. Only 20% of the patients demonstrated specific immune response to M. leprae during ENL. Moreover, high levels of soluble IL-2R (interleukin-2 receptor) were present in 78% of the patients. Circulating anti-neural (anti-ceramide and anti-galactocerebroside antibodies) and anti-mycobacterial antibodies were detected in ENL patients' sera as well, which were not related to the clinical course of disease. Our data suggest that bacterial killing is enhanced during reactions. Emergence of specific immune response to M. leprae and the effective role of TNF-alpha in mediating fragmentation of bacteria still need to be clarified.

Adolescent↗

PubMed2001

Survival and growth of Escherichia coli O157:H7 inoculated onto cut lettuce before or after heating in chlorinated water, followed by storage at 5 or 15 degrees C.

This study determined the effects of mild heat and chlorine treatment followed by storage for up to 18 days at 5 degrees C or 7 days at 15 degrees C on the survival and growth of Escherichia coli O157:H7 inoculated onto fresh-cut iceberg lettuce. The efficacy of treatment with 20 ppm chlorine in killing the pathogen on lettuce at 50 degrees C was determined. Treatment of lettuce with 20 ppm chlorine at either 20 or 50 degrees C did not result in significantly greater reductions in populations of E. coli O157:H7 compared to respective treatments in water without chlorine. The pathogen steadily decreased in viability on treated lettuce throughout subsequent storage at 5 degrees C for 18 days. The population increased by 2.3 to 3.2 log10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage at 15 degrees C. At 4 and 7 days, significantly (alpha = 0.05) higher populations were reached on lettuce that had been treated at 50 degrees C, compared to respective samples that had been treated at 20 degrees C, regardless of the presence of 20 ppm chlorine in the treatment water. Treatment of lettuce with 20 ppm chlorine at 50 or 20 degrees C before or after inoculation with E. coli O157:H7 did not have a marked influence on behavior of the pathogen during subsequent storage at 5 or 15 degrees C.

Chlorine↗

PubMed2003

Fate of salmonellae in citrus oils and aqueous aroma.

The manufacture of orange juice sometimes involves the use of flavor fractions recovered from oranges. The impact of such flavor fractions on Salmonella viability was investigated. A five-strain cocktail of salmonellae was challenged with a singlefold cold-pressed peel oil (CPO), a fivefold CPO, a terpeneless CPO, and an aqueous orange aroma stored at 4 and 25 degrees C. The results obtained in this study indicate that the test compounds possess substantial antimicrobial activity and can cause population reductions larger than the 5-log10 performance standard required by the U.S. Food and Drug Administration's juice hazard analysis critical control point rule (21 CFR 120). The times required to achieve 5-log10 reductions in Salmonella populations ranged from 0.03 to 42.8 h. In general, levels of antimicrobial activity for the test substances were in the following order: terpeneless CPO > five-fold CPO > single-fold CPO > aqueous aroma.

Adaptation, Physiological↗

PubMed2006

Effect of horticultural waste composting on infected plant residues with pathogenic bacteria and fungi: integrated and localized sanitation.

The aim of this work was to study the effect of composting on the viability of plant pathogenic fungi and bacteria. The research consisted of pilot-scale composting of horticultural waste in compost windrows. Studies were carried out on vegetable residues infected with plant pathogenic microorganisms included by either integrated or localized infection. In the first case, the plant pathogen viability was investigated when infected material was mixed throughout compost, while the localized infection was used to study the effect of the composting process on plant waste spot-inoculated with pathogenic microorganisms. Results for localized sanitation showed the total elimination of all tested phytopathogens between 48 and 120 h after composting began. In this case significant differences were observed in relation to 9 different zones in the pile. The disappearance of these microorganisms was similar when all plant waste included in the windrow was infected (integrated infection). Additionally, the results obtained confirmed that the bacteria showed a greater capacity to persist during composting than the fungi. Composting is therefore considered a useful method for recycling horticultural waste and eliminating phytopathogenic bacteria and fungi that inhabit this kind of residue.

Agriculture↗

PubMed1999

Total counts, culturable and viable, and non-culturable microflora of a French mineral water: a case study.

The changes in bacterial counts during the storage of a natural mineral water from a French spring were studied. Samples were taken from the spring and the bottling line. Viable cultivable (VC) bacteria were counted on R2A medium. Total counts, viable and dead bacteria were counted using the LIVE/DEAD Bac Light VIABILITY kit and epifluorescence microscopy. Viable but non-cultivable (VNC) bacteria were estimated by difference between viable and VC counts. Isolates were clustered by phenotype. The microflora in the spring water increased from < 10-3 x 10(5) bacteria ml-1 after 6 d in storage and then stabilized. Mechanical bottling increased the allochthonous bacteria in the water that stabilized at 10(5) bacteria ml-1. Maximal growth is controlled by the low concentration of nutrients in the mineral water and the lysis of dead cells. The allochthonous bacteria came from the aquifer and colonized the filling line. The changes in the VC and VNC populations showed that the bacteria used starvation-survival and entry into the VNC state to adapt to the bottling stress and the enclosed oligotrophic environment.

Beverages↗

PubMed1984

Products of activated mononuclear cells modulate accumulation of collagen by normal dermal and scleroderma fibroblasts in culture.

Supernatants of human mononuclear cells activated with concanavalin A contain soluble factors that modulate accumulation of collagen in human dermal fibroblasts in culture. The supernatants decreased collagen accumulation by 67% in normal fibroblast lines and by greater than 80% in lines established from skin of patients with scleroderma. Collagen was measured by incorporation of 3H-proline into collagenase-sensitive protein. The inhibitory activity of collagen was optimal after a 24-hour incubation of confluent fibroblast monolayers with unfractionated mononuclear cell supernatants. Kinetic studies of this response showed a delay in accumulation of collagen-sensitive protein in supernatant-treated cultures. The 3H-thymidine uptake and fibroblast cell count and viability were only minimally altered. Noncollagen protein was inhibited by 30% to 40%. The presence of serum in fibroblast cultures did not affect this activity. The inhibitory factors were produced by purified T-lymphocyte subpopulations. Supernatant inhibitory activity was present after removal of monocytes from mononuclear cell cultures. By gel filtration on Sephadex G-100, active supernatants fractionated into at least three peaks of activity with molecular weight of 50,000, 30,000, and 15,000 daltons. Interleukin 1 might be one of the factors in mononuclear cell supernatants that modulates production of collagenase-sensitive protein in human dermal fibroblasts. Factors modulating collagen-sensitive protein are important in processes leading to excessive accumulation of the connective tissue and fibrosis in certain diseases.

Cells, Cultured↗

PubMed1982

Fine structure and function of isolated gonadotropic cells as revealed from pituitaries of immature rainbow trout, Salmo gairdneri, by means of a new enzymatic dispersion technique.

A procedure has been developed for dissociating pituitary glands of juvenile rainbow trout, Salmo gairdneri, producing a preparation of single dispersed pituitary cells in which morphological and functional integrity is preserved. The pituitaries are dispersed by sequential treatment with 0.1% collagenase, 0.04% ethylene-diamine-tetra-acetic acid (EDTA) and 0.125% dispase. The cell yield is 0.3--0.35 x 10(6) cells per pituitary with a cell viability percentage of 95 +/- 1% and single cell percentage of 87 +/- 4%. The isolated cells are kept in a suspension system and the gonadotropic cells are identified by the double antibody immuno-enzyme-cytochemical technique using anti-carp-beta-gonadotropin as first antibody. Secretory activity is estimated by measuring the gonadotropin content in cells and culture media by radioimmunoassay. Isolated cells show an autonomy of gonadotropin secretion. 17 alpha-Methyltestosterone both in vivo and in vitro stimulates the production of gonadotropin in the cells and seems to inhibit its release from the cells. It is concluded that this in vitro system can be used as a model for studying the control of gonadotropic cells in juvenile rainbow trout.

Animals↗

PubMed2004

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

Cell Membrane Permeability↗

PubMed2006

In vivo survival of teicoplanin-resistant Staphylococcus aureus and fitness cost of teicoplanin resistance.

Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N-acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10(4) CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N-acetylglucosamine uptake, and increased hemolysis; however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.

Animals↗

PubMed1978

Effect of a staphylococcin on Neisseria gonorrhoeae.

Phage group 2 staphylococcal strain UT0002 contains a large 56S virulence plasmid with genes that code for both exfoliative toxin and a specific staphylococcin termed Bac R(1). Four penicillinase-producing strains and three penicillin-susceptible strains of Neisseria gonorrhoeae were killed by Bac R(1). After 30 min of growth of the penicillin-resistant TR1 strain in 62.5 arbitrary units of Bac R(1) per ml, loss of viability was approximately 90%, and, after 5 h, an approximately 99.99% loss of viability was observed. Lysis did not accompany cell death, and 84% of the Bac R(1) added to the growth medium was adsorbed to the gonococcal cells. The extracellular supernatant fluid from a substrain of staphylococcal strain UT0002 cured of the plasmid for Bac R(1) production had no lethal effect on the gonococcal strains. Bac R(1) was also shown to have bactericidal activity against an L-form of N. meningitidis, indicating that the outer envelope of a neisserial cell is not needed for bacteriocin activity. Ten different normal human sera were unable to neutralize Bac R(1) activity. The bacteriocin lacks adsorption specificity. It binds to but does not kill Escherichia coli cells, indicating that the cell envelope of gram-negative organisms can provide protection against the staphylococcin.

Absorption↗

PubMed1999

A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of alpha-amylase.

A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse alpha-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse alpha-amylase, we observed that transformed colonies grew more quickly and secreted more alpha-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse alpha-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system.

Animals↗

PubMed1996

Structure and mode of action of kikA, a genetic region lethal to Klebsiella oxytoca and associated with conjugative antibiotic-resistance plasmids of the IncN group.

Transmission of conjugative plasmids of the IncN group into Klebsiella oxytoca, but not into Escherichia coli, results in the marked reduction of viability of the recipients. In the plasmid pCU1 a 500-bp locus called kikA has the major role in determining this phenotype. Expression of two open reading frames (orf104 and orf70) is required for the Kik+ phenotype and they can function in cis or in trans. orf104 encodes a 8.9-kDa soluble protein which is translocated into the periplasm. orf70 encodes a 7.6-kDa soluble protein which is found in the cytoplasm. The expression of the kikA region from its natural promoter(s) is positively regulated at the level of transcription by an additional plasmid locus which is located within the tra region. Further experiments show that the action of kikA causes reversible growth inhibition but does not affect cellular respiration and does not induce any morphological changes of the host cell.

Amino Acid Sequence↗

PubMed1993

The effects of modified atmospheres on the growth of psychrotrophic pseudomonads on a surface in a model system.

Atmospheres containing concentrations of CO2 as low as 20% (balance nitrogen) inhibited the growth of Pseudomonas fluorescens and Pseudomonas putida on the surface of buffered Brain Heart Infusion agar plates, pH 6.8, incubated at 5 or 15 degrees C in flexible packages. The modified atmospheres decreased the growth rates and reduced the populations attained at the end of the exponential phase of growth, but had no substantial effect on the lag phase. P. fluorescens was less tolerant of CO2 than P. putida. The inhibitory effect of CO2 increased with its concentration and inhibition was greater at 5 than at 15 degrees C. Growth occurred in packages flushed with 20, 40 and 100% CO2 and 100% N2 at 15 degrees C and 20 and 40% CO2 and 100% N2 at 5 degrees C. The residual O2 concentration in the packages after flushing was 0.2-0.5%. Storage of pseudomonads in CO2 under conditions that prevented growth (e.g., 100% CO2, 5 degrees C) did not cause substantial loss of viability. There was no detectable residual effect of CO2. If cultures were incubated in air after storage for up to 70 days in CO2-containing atmospheres which prevented growth, the subsequent growth curve did not differ noticeably from that observed when plates were incubated in air immediately after inoculation. When cultures in the exponential or stationary phases of growth in modified atmospheres were transferred to air, growth rates increased quickly to rates similar to those observed in air and the final populations observed in air were attained. A reduction in the pH of the medium to 5.5 substantially increased the inhibitory effect of CO2. At 5 degrees C and pH 5.5, substantial growth of P. fluorescens was not observed in any of the CO2 concentrations tested, nor in 40 or 100% CO2 for P. putida.

Air↗