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.

Statistical study of the relationship between free chlorine levels and bacteriological checks on systems in the Paris area.

The majority of French cities are supplied with treated surface water. The primary factor in determining the quality of this water concerns disinfection to ensure that the system is not contaminated with parasites, coliforms and streptococci; some of these organisms, especially coliforms, are capable of multiplying within the system. The presence of bacteria indicative of faecal contamination in water samples, which contains a significant amount of free chlorine, has revealed a possible link between the two. Based upon sampling points of the Parisian system (and in the suburbs for purposes of comparison), we show the existence of a linear relationship between the results of analyses for coliforms on a given day, D, and the content of free chlorine in the water on days D-2 and D-3. Subsequent ground tests confirm the explanations proposed regarding these phenomena.

[Evolution of isolation and identification techniques of waterborne viruses]. / Evolution des techniques d'isolement et d'identification des virus des eaux.

The paralytic potential of the poliovirus was recognized as early as the 14th century B.C. as illustrated in Egyptian art. But it is only after the four last decades that methods for their concentration from water and their identification were performed. Among several of them the adsorption-elution method was retained. Nevertheless two important barriers had to be ran-over. The first one was the concentration-elution steps on different materials which had to be improved. The second one was the typing method which had to move from particle by particle identification to entire viral population. Despite of these advances only a few cytopathogenic serotypes were found. The reverse transcriptase-polymerase chain reaction with its far more wide spectrum allows the fast and direct identification of viral nucleic acids (or their fragments) of almost all viruses, cytopathogenic or not. With this method elevated amounts of drinking water samples were found positive for several non cytopathogenic viruses. The sanitary significance of these results has still to be proved.

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.

Cytotoxicological safety assessment of papers and boards used for food packaging.

Because of the lack of information about the possible transfer of toxic compounds from papers and boards to food, the overall cytotoxicity induced by six papers and 15 boards was investigated from water extracts prepared according to the European prestandard. Cytotoxicity measurements were based on RNA synthesis rate of human HeLa S3 cells. The tested virgin and recycled papers and boards were differentiated and classified according to their cytotoxicological quality, which ranged from absence of any cytotoxic effect to severe inhibition of RNA synthesis rate. The cytotoxicity level also varied according to the total amount of compounds detected by gas chromatography. No correlation was found between cytotoxicity and endotoxins contained in the samples. No significant difference in cytotoxicity was observed between the papers and boards produced from virgin fibres and from recycled fibres. Moreover, the products obtained from chemical pulp showed lower cytotoxicity than the products based on mechanical pulp. More generally, the cytotoxicological approach is promising for monitoring paper/board treatment-induced problems. Further work is required to assess a modified standard procedure for the preparation of water extracts specifically adapted to paper/board.

High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain.

A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km. The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms. The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase. As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid. By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector. Efficiency of the transposition process was 0.58%. This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.

[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.

Time series analysis on chlorides, nitrates, ammonium and dissolved oxygen concentrations in the Seine river near Paris.

Graphical and statistical time series techniques have been used to analyze the trends and specified time changes, in a 90-year record of annual average value of Seine river water quality data. The information obtained may be associated with some socio-economic variables, such as industrial or agricultural development, urban increase and wastewater discharge around or upstream of the measure station. Such a study may now be applied to more rural stations in order to compare the evolution of water quality and, perhaps, historical monthly average values to evaluate the seasonality effect on annual trends.

[Lead in drinking water, determination of its concentration and effects of new recommendations of the World Health Organization (WHO) on public and private networks management]. / Le plomb dans les eaux d'alimentation, détermination de sa concentration et incidence des nouvelles recommandations de l'Organisation Mondiale de la Santé (OMS) sur la gestion des réseaux de distribution, publics et privés.

In 1993, the World Health Organization (WHO) has given a guideline value of 10 microgram/l for lead in drinking water, a phased approach should lead to a temporary parametric value of 25 micrograms/l within 5 years the final concentration value of 10 micrograms/l being achieved after 15 years. So far the current European Community Directive 80/778 and the French decree 89/3 stipulate a Maximum Admissible Concentration (MAC) for lead of 50 micrograms/l. In a first step we studied the mechanisms of plumbosolvency in corrosive and scaling water. In the first case we have shown that simple oxidative corrosion of lead pipes forms a coating of lead carbonate and hydroxicarbonate on the inside wall of the pipe but "plumbosolvent" waters can dissolve those products, although at a lower level, resulting in a rather high lead concentration. In the case of scaling waters there is a co-precipitation of insoluble calcium carbonate but only on the microcathodics zones of the lead pipe. As this precipitate is poorly cohesive and does not cover the entire surface of the pipe its oxidative corrosion can proceed. In a second step we have shown the major importance of sampling for the determination of lead concentration in drinking water. We therefore compared random day time sampling, first draw and flushed samplings and composite proportional sampling. Only this last method gave a reasonably accurate idea of lead's amounts ingested by drinking water's consumers. The control of corrosion in lead-containing materials involves two successive steps: the reduction of lead concentration to 25 micrograms/l within five years and the compliance with the final 10 micrograms/l concentration 15 years later. The first step consists in water treatments such as pH increase, adjustment of alkalinity and addition of orthophospates. But available data suggest that it is unlikely that lead concentration could be reduced consistently to below 10 micrograms/l by avalable water treatment methods alone but it would enable to match the parametric 25 micrograms/l value in the great majority of cases. Therefore, to unable compliance with the 10 micrograms/l parametric value, it will be necessary to replace all the internal plumbing and supply lead pipes (70,000 buildings for Paris only). Data for materials able to replace lead such as plastic pipes are not yet complete and an currently under investigations. Although the United States Environmental Protection Agency have suggested in its 1988 report on air quality criteria for lead report (EPA 600/8-33-028 aF-dF) that each 1 microgram/l of lead in water can lead to an increase of blood lead levels of approximately 0.2 micrograms/l for a child, the data are still uncertain. The considerable cost of these works (143 billion of french francs for France and 347 billions of french francs for Europe), unrelated to any important Public Health problems, arises an ethical problem which has to be considered in view of many others letal illnesses such as heart and circulatory diseases, cancer and AIDS.

[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.

[Continuous detection of poliovirus in public water supplies]. / Détection en continu du poliovirus dans des eaux de distribution publique.

Continuous virological control can be carried out when using glass wool as an adsorption support for virus concentration. In the described conditions more than 70% of the Poliovirus population in water pipe derivations can be detected.

[Inhibition of poliovirus replication by an extract of Matricaria chamomilla (L)]. / Inhibition de la replication du Poliovirus par un extrait de Matricaria chamomilla (L).

Hydroalcoholic extract of Matricaria chamomilla added during the early stage of Poliovirus development inhibits cellular and viral RNA synthesis. This inhibition is partially reversible.

Preformed magnesium hydroxide precipitate for second-step concentration of enteroviruses from drinking and surface waters.

A method is described for the second-step concentration of viruses from large volumes of drinking and surface waters. Seeded viruses present in the first eluate, performed with 50 mM glycine buffer, pH 11.5, were adsorbed on a preformed magnesium hydroxide precipitate. After low-speed centrifugation they were desorbed and adjusted to pH 7 with McIlvaine citrate-phosphate buffer. In these experimental conditions 90% of the viruses present in the 300-mL first eluate were reconcentrated in a final volume of 40 mL. The recovery efficiency was independent of either virus concentration or water quality.