Differential persistence of F-specific RNA phage subgroups hinders their use as single tracers for faecal source tracking in surface water.

The four subgroups of F-specific RNA bacteriophages (I-IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated. The proportions of the F-specific RNA bacteriophage subgroups were measured in wastewaters, after inactivation in surface waters or after wastewater treatment and in mixtures of wastewater of human and animal origin. Our results indicate that phage groups differ in their persistence in the environment and to different disinfecting treatments. The greater survival of subgroups I and II in treated samples hinders the interpretation of results obtained with F-specific RNA bacteriophages. The phages of subgroups III and IV were the least resistant to all treatments. These results should be considered when using genotypes of F-specific RNA as sole tracers for faecal source tracking.

Evaluation of Escherichia coli host strain CB390 for simultaneous detection of somatic and F-specific coliphages.

Escherichia coli WG5, the strain recommended by the International Organization for Standardization (ISO) to detect somatic coliphages, was transformed to F(+) by introducing the plasmid Famp, which rendered it capable of simultaneously detecting both somatic and F-specific coliphages. Indeed, this strain, CB390, proved as effective in detecting similar numbers of phages as the sum of somatic and F-specific bacteriophages detected by the host strains recommended by both the ISO and the U.S. Environmental Protection Agency standardized methods.

A membrane-based quantitative carrier test to assess the virucidal activity of disinfectants and persistence of viruses on porous fomites.

A membrane-based quantitative carrier test method to assess the virucidal activity of disinfectants and the persistence of viruses on fomites under different environmental conditions is described. The method is based on the inactivation of the virus adsorbed to cellulose ester membranes followed by the direct enumeration of the viruses surviving the treatment without the need of an elution step. The method was suitable for four different human enteroviruses tested. Experiments comparing the infectivity loss of human enteroviruses in suspension or adsorbed to the filters after treatment with chlorine and glutaraldehyde showed that the human enteroviruses tested suffered significantly greater log10 reductions when suspended than when adsorbed. Significant differences in the effect of the disinfectants on the various human enteroviruses tested were also observed. Moreover, the procedure allowed determining the inactivation of viruses on fomites under different environmental conditions. Low temperatures and high relative humidities favored the survival of human enteroviruses. Also, viruses adsorbed to the membranes retained their infectivity frozen at -70 degrees C for more than 1 year, thus providing the possibility of preparing very simple reference materials for testing virucidal activities of antiseptics and disinfectants.

Enteroviruses and bacteriophages in bathing waters.

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting Bacteroides thetaiotaomicron were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting Bacteroides persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters.

Double-layer plaque assay for quantification of enteroviruses.

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay >or=suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters >or= most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2'-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.

A method to maintain mammalian cells for days alive at 4 degrees C.

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 degrees C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.

Bacterial host strains that support replication of somatic coliphages.

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log10 fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible.

Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage.

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.

Counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters as a simple method for counting viruses in raw sewage and sewage effluents.

Using the VIRMETADEN (acronym derived from virus adsorption enumeration) method to count cytopathogenic viruses adsorbed to cellulose nitrate membrane filters from prefiltered and decontaminated sewage samples was shown to be feasible. The numbers of naturally occurring enteroviruses recovered by the VIRMETADEN method were significantly higher than those obtained by standard plaque assay. For prefiltration of sewage samples, low protein binding polyvinylidene fluoride (PVDF) membranes of 0.65 and 5 microm pore size proved to be at least as good as prefilters used previously, both in the volume that could be filtered and in the low virus retention. Regarding decontamination, 0.22 microm pore size PVDF membranes were as good as chloroform treatment. Alternatively, decontamination with 0.05% phenol followed by filtration through 0.65 microm PVDF membrane filters proved to be an excellent method for clarifying and decontaminating the sample prior to enumerating viruses using the VIRMETADEN method. It allows good recovery and greater decontaminated sample volumes. The application of VIRMETADEN to decontaminated raw sewage and secondary effluent gave recoveries of approximately 100% of vaccinal polioviruses seeded in sewage. The advantage of this approach is that the same method is applicable to sewage affluents and effluents with a simple and fast method, which implies lower material and labour costs.