Effect of gamma irradiation on bacteriophages used as viral indicators.

This study aimed to examine the susceptibility of indicator bacteriophages towards γ-radiation to evaluate their appropriateness as viral indicators for water quality control. The effects of γ-radiation on naturally occurring somatic coliphages, F-specific coliphages and Escherichia coli were examined in raw sewage and sewage sludge. As well, the effects of radiation on bacteriophages ΦX174 and MS2, and E. coli all grown in the laboratory and seeded in distilled water, autoclaved raw sewage and a 1% peptone solution were evaluated. The inactivation of E. coli was fairly similar in all matrices. In contrast, inactivation of bacteriophages was significantly greater in distilled water than in the other matrices. These results showed the great influence of the matrix characteristics on virus inactivation. Somatic coliphages in raw sewage and sewage sludge and ΦX174 in autoclaved sewage were inactivated similarly and were far more resistant than F-specific coliphages, MS2 and E. coli. As well, F-specific RNA bacteriophages in raw sewage and sewage sludge and MS2 in autoclaved sewage were inactivated similarly and were more resistant than E. coli. In contrast, MS2 was more susceptible to γ-radiation than E. coli in distilled water. Our results showed that ΦX174 is a suitable indicator for estimating virus inactivation by γ-irradiation and corroborate the use of somatic coliphages to survey the viral quality of treated water and sludges.

Simulated sunlight inactivation of norovirus and FRNA bacteriophage in seawater.

AIMS: To investigate norovirus (NoV) and F-specific RNA (FRNA) bacteriophage inactivation in seawater under simulated sunlight and temperature conditions representative of summer (235 W m(-2) ; 17°C) and winter (56 W m(-2) ; 10°C) conditions in Ireland. METHODS AND RESULTS: Inactivation experiments were carried out using a collimated beam of simulated sunlight and 100 ml of filtered seawater seeded with virus under controlled temperature conditions. NoV concentrations were determined using RT-qPCR, and FRNA bacteriophage concentrations were determined using RT-qPCR and by plaque assay. For all virus types, the fluence required to achieve a 90% reduction in detectable viruses (S90 value) using RT-qPCR was not significantly different between summer and winter conditions. S90 values for FRNA bacteriophage determined by plaque assay were significantly less than those determined by RT-qPCR. Unlike S90 values determined by RT-qPCR, a significant difference existed between summer and winter S90 values for infectious FRNA bacteriophage. CONCLUSIONS: This study demonstrated that RT-qPCR significantly overestimates the survival of infectious virus and is therefore unsuitable for determining the inactivation rates of viruses in seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study provide initial data on the inactivation of NoV and FRNA bacteriophage in seawater under representative summer and winter conditions and will be of interest to shellfish and water management agencies alike.

UV disinfection of RBC-treated light greywater effluent: kinetics, survival and regrowth of selected microorganisms.

The microbial quality of raw greywater was found to be much better than that of municipal wastewater, with 1.6 x 10(7)cfu ml(-1) heterotrophic plate count (HPC), and 3.8 x 10(4), 9.9 x 10(3), 3.3 x 10(3) and 4.6 x 10(0)cfu 100 ml(-1) faecal coliforms (FC), Staphylococcus aureus sp., Pseudomonas aeruginosa sp. and Clostridium perfringes sp., respectively. Further, three viral indicators monitored (somatic phage, host: Escherichia coli CN(13) and F-RNA phages, hosts: E. coli F+(amp), E. coli K12) were not present in raw greywater. The greywater was treated by an RBC followed by sedimentation. The treatment removed two orders of magnitude of all bacteria. UV disinfection kinetics, survival and regrowth of HPC, FC, P. aeruginosa sp. and S. aureus sp. were examined. At doses up to 69 mW s cm(-2) FC were found to be the most resistant bacteria, followed by HPC, P. aeruginosa sp. and S. aureus sp. (inactivation rate coefficients: 0.0687, 0.113, 0.129 and 0.201 cm2 mW(-1)s(-1), respectively). At higher doses (69-439 mW s cm(-2)) all but HPC (which exhibited a tailing curve) were completely eliminated. Microscopic examination showed that FC self-aggregate in the greywater effluent. This provides FC an advantage at low doses, since the concentration of suspended matter (that can provide shelter from UV radiation) in the effluent was very low. FC, P. aeruginosa sp. and S. aureus sp. did not exhibit regrowth up to 6h after exposure to increasing UV doses (19-439 mW s cm(-2)). HPC regrowth was proven to be statistically significant in un-disinfected effluent and after irradiation with high UV doses (147 and 439 mW s cm(-2)). At these doses regrowth resulted from growth of UV-resistant bacteria due to decreased competition with other bacteria eliminated by the irradiation.

Virus removal in a pilot-scale 'advanced' pond system as indicated by somatic and F-RNA bacteriophages.

Advanced pond systems (APS), incorporating high-rate ponds, algal settling ponds, and maturation ponds, typically achieve better and more consistent disinfection as indicated by Escherichia coli than conventional waste stabilisation ponds. To see whether this superior disinfection extends also to enteric viruses, we studied the removal of somatic phages ('model' viruses) in a pilot-scale APS treating sewage. Measurements through the three aerobic stages of the APS showed fairly good removal of somatic phage in the summer months (2.2 log reduction), but much less effective removal in winter (0.45 log reduction), whereas E. coli was removed efficiently (> 4 logs) in both seasons. A very steep depth-gradient of sunlight inactivation of somatic phage in APS pond waters (confined in silica test tubes) is consistent with inactivation mainly by solar UVB wavelengths. Data for F-RNA phage suggests involvement of longer UV wavelengths. These findings imply that efficiency of virus removal in APS will vary seasonally with variation in solar UV radiation.

Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.

Methylene blue and rose bengal photoinactivation of RNA bacteriophages: comparative studies of 8-oxoguanine formation in isolated RNA.

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.

Destruction of bacterial viruses in serum by heat and radiation under conditions that sustain the ability of serum to support growth of cells in suspended culture.

Methods for inactivating bacterial viruses in serum were developed through the use of heat and ionizing radiation, and the effects of these treatments on the growth rates of cultured cells were tested. Viruses chosen for this study were the radiation-resistant bacteriphage f2 and heat-resistant phage T4. The viabilities of these phages were reduced more than 2 and 4 orders of magnitude, respectively, by a treatment at 60 degrees C for 30 min followed by 420 krads of ionizing radiation. Simultaneous application of heat and radiation caused a considerably greater reduction in viability of both phages in serum, but also caused a significant decrease in the growth rates of L cells in medium supplemented with serum treated in this manner. Treatment of serum with these same doses but given in the sequential fashion of heat followed by radiation caused little or no change in the growth rates of L cells. Finally, it was found that simultaneous treatment of serum with these doses of heat and radiation had little effect on the growth rates of either HeLa or Chinese hamster cells.