Methods for biofilm analysis on silicone tubing of dialysis machines.

We describe an analytical protocol to study biofilms that develop inside silicone tubing of dialysis machines. This protocol has been set up with the help of a dynamic testing device reproducing dialysis conditions. The methodology includes direct microscopic observation, biofilm removal with an original mechanical biofilm scraper, quantitative analysis with culturable and total bacteria counting, and endotoxin level measurement using the LAL chromogenic kinetic assay. The analytical protocol has been assessed on 13 different clinical tubing samples. Most samples were contaminated by adherent cells and the thickest biofilms were found at the connection between the dialysis water distribution loop and the dialysis machine. The less contaminated samples had been removed from dialysis machines that were decontaminated with citric acid and autoclaving, showing the importance of the decontamination procedure for the prevention of biofilm development. This article shows that easy, rapid, reproducible, and economical methods are applicable for a routine analysis of biofilms that develop on dialysis systems and should be included in the regular control of the microbiological quality of dialysis liquids.

Biofilm removal from silicone tubing: an assessment of the efficacy of dialysis machine decontamination procedures using an in vitro model.

The aim of this study was to assess the efficacy of 21 decontamination procedures, for the removal of a multispecies biofilm. Experiments were performed on five-day-old biofilms grown inside silicone tubing, using a reactor system that mimics a dialysis machine. The treatments were tested on 5 cm tubing samples. Effects of treatment were measured using direct microscopy following staining. Bacterial viability and endotoxin removal were determined using conventional microbiological methods following biofilm detachment by scraping. The 21 procedures were classified into four groups based on the amount of biofilm removed. The most effective treatment was an acid pre-treatment, followed by use of a concentrated bleach solution. Acid pre-treatment removes calcium and magnesium carbonate crystals that are always found in dialysis biofilms. Treatments performed at high temperature did not increase the efficacy of biofilm removal. Most treatments caused at least a 10(5)-fold reduction in bacterial viability with a few resulting in complete kill. Autoclaved and bleach-treated samples gave the best results for viability reduction, with both treatments providing an equally effective and complete kill. In addition, autoclaving led to a significant decrease in endotoxin level (removal of 99.99%).

Cytotoxicity assessment of chlorinated bacteria in water using the RNA synthesis inhibition method.

Chlorination of drinking water containing organic materials is known to generate toxic by-products. We suggested that such compounds may also be produced by interactions between chlorine and bacteria present in water. To confirm this hypothesis, a method based on RNA synthesis inhibition of HeLa S3 human cells in the presence of toxic compounds was applied. This method is rapid and highly sensitive since the concentration of the samples is not required. Furthermore, it was shown to be a suitable method for measurement of the cytotoxicity of water. Aeromonas hydrophila suspensions, prepared with pyrodistilled water, devoid of any organic material, were chlorinated for a definite contact time. HeLa S3 cells were incubated (20 h, 37 degrees C) in a culture medium prepared with the chlorinated bacteria suspensions. The rate of incorporation of 3H uridine into RNA was used as a measure of RNA synthesis and was evaluated in the presence and absence of chlorinated bacteria suspension. This study showed that chlorinated bacteria suspensions are cytotoxic. We observed that 0.22 microm filters retain cytotoxic compounds but 0.45 microm filters did not. Chlorine concentration and bacteria level influence the cytotoxicity. First, the toxicity level increases with chlorine concentration, then it decreases when chlorine concentration is too high. On another hand, a dose effect relationship between bacteria concentration and cytotoxicity was established.

[Antiviral activity of hydroalcoholic extract from Haemanthus albiflos on the Moloney murine leukemia virus and the human immunodeficiency virus]. / Activité antivirale d'un extrait hydro-alcoolique d'Haemanthus albiflos sur le virus de la leucémie murine de Moloney et le virus du syndrome de l'immunodéficience acquise.

We have used a biological test on the microplates of cellular cultures in order to investigate the toxicity and the antiviral properties against different viruses: defective Moloney Murine Leukemia virus (MoMLV) derived from the SVX shuttle and expressing resistance to the G418 antimitotic, and Human Immunodeficiency Virus (HIV) of a hydroalcoholic extract from Haemanthus albiflos (Amaryllidacae). The toxicity was assessed through coloric test evaluation of fixed cells stained with crystal violet. In a population of NIH 3T3 cells (Fibroblasts mouse), the toxicity found with 2, 7, 14 and 28 microliters/ml of lyophilisat extract corresponding at: 0.23, 0.81, 1.62 and 3.24 mg of plant dry, was 32, 50, 63 and 70% respectively. With regards to the antiviral properties, the plant extracts showed an inhibition of 88% on the formation of G418 resistant 3T3 clones. The assay on HIV infected lymphotic cells (P4) showed an IC50 of 4 microliters/ml for this extract plant. Therefore, the toxic effect was similar to the antiviral response.

[Use of cell culture for the research of cytotoxic and antiviral effects of a natural extract of Amaryllidaceae]. / Utilisation des cultures cellulaires pour la recherche des effets cytotoxiques et antiviraux d'un extrait naturel d'Amaryllidaceae.

The use of tissue culture for evaluation of antiviral agents can provide rapid information on the toxicity induced by drugs. Toxicity is assessed through 4 different tests: observation through a light microscope, colorimetric evaluation of living cells stained with MTT (Methyl Thiazol Tetrazolium), colorimetric evaluation of fixed cells stained with crystal violet, inhibition of incorporation of radioactive labelled precursors specific to the synthesis under study. These tests allowing evaluation of effects on cell morphological changes (1), of modifications of mitochondrial and enzymatic activities in the cytoplasm (2), of effects on cell growth (3) and on their major synthesis [ADN, ARN, proteins] (4). This design of experiments has been applied to an hydroalcoolic extract from Haemanthus albiflos (Amaryllidaceae) tested for its antiviral properties towards Poliovirus type 1 propagated on monkey kidney cells line (MA 104). The maximum tolerated dose by the cell and the inhibition of virus replication were determined according to tests 2 and 3. The sensitive step of the virus replication cycle was investigated using test 4. Concentration of 7 microliters/ml plant extract showed: 20% cytotoxicity (MTT test). At 7 microliters/ml plant extract the inhibition of replication virus is 4.5 log units (microplates assay) and inhibition of proteins viral synthesis is 97% compared with the control.

[Study of antiviral action of total alkaloids from Haemanthus albiflos]. / Etude de l'action antivirale des alcaloïdes totaux d'Haemanthus albiflos.

Investigations were undertaken on the antiviral action level of an alkaloïd extract from Haemanthus albiflos bulb, earlier reported as efficient against RNA viruses. Rotavirus propagated on MA 104 cells with different concentrations of the extract was used in the assays. Incidence on cellular and viral RNA synthesis was evaluated by measuring the radioactivity incorporated using labelled precursors. An inhibition of 42% and 79% of the cellular RNA synthesis was observed when respectively 25 microliters/ml and 50 microliters/ml concentrations of the alkaloïd extract were tested. After 20 h incubation a decrease of the viral RNA synthesis was observed. It was of 46%, 36% and 27% compared to the control when respectively 25 microliters/ml, 50 microliters/ml and 100 microliters/ml concentrations of the extract were tested. Besides, the maximum viral production was delayed parallelly to the increase of the extract concentration. A similar viral synthesis inhibition was obtained after only 4 hours incubation suggesting that the extract interfere in the early events of the viral cycle.

[Plants as a source of research in antiviral activity. Example of the Haemanthus albiflos bulb]. / Les plantes comme source de recherche d'activité antivirale. Exemple du bulbe d'haemanthus albiflos.

The antiviral potency of an hydroalcoholic extract from Haemanthus albiflos (AMARYLLIDACEAE) bulb was investigated. Experimentations were conducted on continuous cell lines (BGM, MA 104, Hep 2) seeded in microplates. Three viruses from the RNA group (Poliovirus type I, Vesicular Stomatitis Virus type 11 and Simian Rotavirus SA 11) and two from the DNA group (Adenovirus type 5, Herpes Simplex Virus type 1) were tested. Important reduction in yield of viral infectivity was observed with the RNA group (respectively 6,4 and 4,5 logarithmic units order of magnitude).

[Antiviral effect of Haemanthus albiflos leaves extract on herpes virus, adenovirus, vesicular stomatitis virus, rotavirus and poliovirus]. / Effet antiviral d'un extrait de feuilles d'Haemanthus albiflos sur le virus de l'herpès, l'Adénovirus, le virus de la stomatite vésiculeuse, le Rotavirus et le Poliovirus.

An hydro-alcoholic extract from Haemanthus albiflos leaves (Amaryllidaceae) was tested for its potential antiviral activity against two DNA viruses: herpes simplex virus type I, Adenovirus type 5 and three RNA viruses: poliovirus type I, vesicular stomatitis virus, simian Rotavirus SA 11. Positive results were obtained against herpes virus and all the RNA viruses tested.