Coliphages as Indicators for the Microbial Quality of Treated Wastewater Effluents.

Wastewater effluents are a reliable water source for non-potable water reuse including unrestricted crop irrigation in arid regions suffering from water scarcity. This study was performed to develop and optimize a procedure to concentrate coliphages from 100 L of treated effluent. Moreover, the reduction of coliphages by filtration and disinfection by either chlorine or UV was compared with that of fecal coliform (FC). The adsorption efficiency of MS2 and Qß coliphages by the NanoCeram filter was similar and reached 99.8%. Elution efficiency of MS2 coliphage from the NanoCeram filters by a solution of 1% NaPP and 0.05 M glycine, pH 9.5, was 74  ±  9.5%. The highest reconcentration efficiency of MS2 and Qß coliphages was obtained with polyethylene glycol (PEG) precipitation and reached 76  ±  28% and 90  ±  11%, respectively. In comparison, the reconcentration efficiency of organic flocculation was 0% and 1.3% for Qß and MS2 coliphages, respectively. The overall recovery efficiency of MS2 coliphages from 100 L tertiary effluent was 57  ±  1.5%. Poor reduction was observed for coliphages compared to FC by filtration and chlorine disinfection although; the reduction of FC, as measured by cultivation, was satisfactory and within the guidelines for unrestricted irrigation. High correlation between the reduction of FC and coliphages was recorded for tertiary effluent disinfected by UV irradiation. Monitoring the microbial quality of tertiary effluent using qPCR for the enumeration of FC was found unsuitable, because DNA levels were unaffected by the treatment processes. The results of this study demonstrated that monitoring the microbial quality of tertiary effluent by FC may not reflect the health risks encountered by the application of these effluents and the addition of coliphages to the monitoring programs may allow for accurate assessment of the health risks introduced by the application of tertiary effluent.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

Solar Disinfection of Viruses in Polyethylene Terephthalate Bottles.

Solar disinfection (SODIS) of drinking water in polyethylene terephthalate (PET) bottles is a simple, efficient point-of-use technique for the inactivation of many bacterial pathogens. In contrast, the efficiency of SODIS against viruses is not well known. In this work, we studied the inactivation of bacteriophages (MS2 and ϕX174) and human viruses (echovirus 11 and adenovirus type 2) by SODIS. We conducted experiments in PET bottles exposed to (simulated) sunlight at different temperatures (15, 22, 26, and 40°C) and in water sources of diverse compositions and origins (India and Switzerland). Good inactivation of MS2 (>6-log inactivation after exposure to a total fluence of 1.34 kJ/cm(2)) was achieved in Swiss tap water at 22°C, while less-efficient inactivation was observed in Indian waters and for echovirus (1.5-log inactivation at the same fluence). The DNA viruses studied, ϕX174 and adenovirus, were resistant to SODIS, and the inactivation observed was equivalent to that occurring in the dark. High temperatures enhanced MS2 inactivation substantially; at 40°C, 3-log inactivation was achieved in Swiss tap water after exposure to a fluence of only 0.18 kJ/cm(2). Overall, our findings demonstrate that SODIS may reduce the load of single-stranded RNA (ssRNA) viruses, such as echoviruses, particularly at high temperatures and in photoreactive matrices. In contrast, complementary measures may be needed to ensure efficient inactivation during SODIS of DNA viruses resistant to oxidation.

Influence of environmental variables in the efficiency of phage therapy in aquaculture.

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.

Persistence of infectious Shiga toxin-encoding bacteriophages after disinfection treatments.

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log10 reduction for an estimated UV dose of 178.2 mJ/cm(2)) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log10 reductions, respectively) and were highly inactivated (3 log10) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.

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.

Assessment of a portable handheld UV light device for the disinfection of viruses and bacteria in water.

Effective individual microbiological water purifiers are needed for consumption of untreated water sources by campers, emergency use, military, and in developing counties. A handheld UV light device was tested to assess if it could meet the virus reduction requirements established by the United State Environmental Protection Agency, National Science Foundation and the World Health Organization. The device was found capable of inactivating at least 4 log10 of poliovirus type 1, rotavirus SA-11 and MS-2 virus in 500 mL volumes of general case test water. But in the presence of high turbidity and organic matter, filtration was necessary to achieve a 4 log10 reduction of the test viruses.

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat.

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23-25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall.

Human virus and bacteriophage inactivation in clear water by simulated sunlight compared to bacteriophage inactivation at a southern California beach.

Few quantitative data exist on human virus inactivation by sunlight and the relationship between human and indicator viruses under sunlit conditions. We investigated the effects of sunlight on human viruses (adenovirus type 2, poliovirus type 3) and bacteriophages (MS2, Q-Beta SP, Fi, M13, PRD1, Phi-X174, and coliphages isolated from Avalon Bay, California). Viruses were inoculated into phosphate buffered saline or seawater, exposed to a laboratory solar simulator for ≤12 h, and enumerated by double agar layer or cell culture to derive first-order inactivation rate constants (k(obs), h(-1)). The viruses most resistant to sunlight were adenovirus type 2 (k(obs)= 0.59 ± 0.04 h(-1)) and bacteriophage MS2 (k(obs)= 0.43 ± 0.02 h(-1)), which suggests MS2 may be a conservative indicator for sunlight resistant human viruses in clear water when sunlight inactivation is the main removal mechanism. Reasonable agreement was observed between somatic coliphage inactivation rates measured in the solar simulator (k(mean) = 1.81 h(-1)) and somatic coliphages measured in the surf zone during a field campaign at Avalon Bay during similar sunlight intensity (k = 0.75 h(-1) at log-RMSE minimum; k(range) = 0.54 h(-1) to >1.88 h(-1); Boehm, A. B. et al. Environ. Sci. Technol. 2009, 43, (21), 8046-8052). Hence, measuring sunlight inactivation rates of viruses in the laboratory can be used to estimate inactivation in the environment under similar sunlight and water quality conditions.

Advanced process of microbiological control of wastewater in combined system of disinfection with UV radiation.

The purpose of this study was to present a methodology with superior efficiency for inactivating pathogenic indicators commonly found in domestic sewage. The adopted method was based on synergistic effect resulting from the introduction of a UV radiation pre-disinfection stage of sewage followed by secondary treatment. A pilot unit was installed in the sewage treatment plant of the University of São Paulo to simulate the combined system in full-scale operational conditions. Its performance was evaluated through microbiological examinations for determining Escherichia coli, total coliforms and coliphages. The application of UV radiation at 5.1 mW/cm(2) for 10 s of exposure in the first disinfection stage was enough to reduce the surviving number of E. coli around 100 times, in comparison to the conventional method. Therefore, based on experimental data, it is possible to conclude that combining treatment and pre-disinfection stage is an effective potential technique to produce effluents with lower degree of contamination by pathogenic organisms.

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.

Effects of UV intensity and water turbidity on microbial indicator inactivation.

The effects of UV intensity and turbidity on selected microbial indicator inactivation were investigated. Results showed that UV disinfection was effective in killing all the selected microbial indicators, the resistance order of the microorganisms was as follows: MS-2 coliphage > Bacillus subtilis > E. coli > Staphylococcus aureus and Candida albicans. UV intensity had influence on the inactivation of all the microorganisms, high UV disinfection efficency was obtained with higher UV intensity. Turbidity had impact on the bacteria inactivation rate, but there was no evidence that turbidity had any negative contribution to MS-2 coliphage. Under the same UV dosage, higher UV intensity could overcome the negative influence of turbidity on UV performance, enhanced microorganism inactivation effect in turbidity water.

Impact of iron particles in groundwater on the UV inactivation of bacteriophages MS2 and T4.

AIMS: To investigate the impact of iron particles in groundwater on the inactivation of two model viruses, bacteriophages MS2 and T4, by 254-nm ultraviolet (UV) light. METHODS AND RESULTS: One-litre samples of groundwater with high iron content (from the Indianapolis Water Company, mean dissolved iron concentration 1.3 mg l(-1)) were stirred vigorously while exposed to air, which oxidized and precipitated the dissolved iron. In parallel samples, ethylenediaminetetra-acetic acid (EDTA) was added to chelate the iron and prevent formation of iron precipitate. The average turbidity in the samples without EDTA (called the 'raw' samples) after 210 min of stirring was 2.7 +/- 0.1 NTU while the average turbidity of the samples containing EDTA (called the 'preserved' samples) was 1.0 +/- 0.1 NTU. 'Raw' and 'preserved' samples containing bacteriophage MS2 were exposed to 254-nm UV light at doses of 20, 40, or 60 mJ (cm(2))(-1), while samples containing bacteriophage T4 were exposed to 2 or 5 mJ (cm(2))(-1), using a low pressure UV collimated beam. The UV inactivation of both phages in the 'raw' groundwater was lower than in the EDTA-'preserved' groundwater to a statistically significant degree (alpha = 0.05), due to the association of phage with the UV-absorbing iron precipitate particles. A phage elution technique confirmed that a large fraction of the phage that survived the UV exposures were particle-associated. CONCLUSIONS: Phages that are associated with iron oxide particles in groundwater are shielded from UV light to a measurable and statistically significant degree at a turbidity level of 2.7 NTU when the phage particle association is induced under experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: While the particle association of the phage in this study was induced experimentally, the findings provide further evidence that certain particles in natural waters and wastewaters (e.g. iron oxide particles) may have the potential to shield viruses from UV light.

Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages.

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Reduction of enteric microbes in flushed swine wastewater treated by a biological aerated filter and UV irradiation.

An aerobic biofilter system was studied to assess its effectiveness for reducing enteric microbial indicators in flushed swine wastewater under different seasonal conditions. A laboratory-scale, low-pressure UV collimated beam apparatus was used to investigate the effectiveness of UV irradiation for inactivating enteric bacteria, coliphages, and bacterial spores in treated and untreated swine wastewater having unfiltered absorbances of 5 to 11 cm(-1) and total suspended solids concentrations of 500 to 1200 mg/L. Fecal coliforms, Escherichia coli, enterococci, somatic coliphages, and male-specific coliphages were reduced by 97 to 99% in the biofilter system when reactor water temperatures were between 23 and 32 degrees C. Salmonella were reduced by 95 to 97% when water temperatures were 17 to 32 degrees C. Of the six microbial indicators studied. Clostridium perfringens spores were typically reduced the least by the biofilter system. At an average absorbed UV irradiation dose of 13 mJ/cm2, maximum reductions of fecal coliforms, E. coli, enterococci, C. perfringens spores, and somatic coliphages in biofilter system effluent were 2.2, 2.1, 1.3, 0.2, and 2.3 log10, respectively. The results of this study show that the aerobic biofilter system can be an effective alternative for treatment of flushed swine waste. Ultraviolet irradiation can be effective for further reducing enteric microbe concentrations in biologically-treated swine waste, as well as in lower quality wastewaters, indicating its general potential for pathogen reductions in low-quality wastewaters intended for beneficial reuse.

Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters.

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.

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.

Influence of nucleotide excision repair of Escherichia coli on radiation-induced mutagenesis of double-stranded M13 DNA.

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).

Mutations and oxidative DNA damage in phage M13mp2 exposed to N-nitrosomorpholine plus near-ultraviolet light.

Previously we reported that a direct-acting mutagen can be formed from N-nitrosomorpholine (NMOR) on exposure to near-ultraviolet light (UVA, 320-400 nm). We have now studied the spectrum of mutations caused by NMOR plus UVA. M13mp2 phages suspended in a sodium phosphate buffer were treated with NMOR under UVA irradiation and Escherichia coli NR9099 was then infected with the phage. Mutations induced in the phage DNA lacZ alpha region were analyzed. The majority (approximately 50%) of the induced sequence changes were G to T transversions. This suggested that modifications in guanine residues were responsible for these transversions. We explored the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) in the DNA. When the phage were treated with NMOR plus UVA, 8-oxodG/dG in DNA increased up to 12-fold over the value in untreated control. When a mutM-deficient mutant of E. coli CSH50 was used as the host, the mutation level was higher than that observed with CSH50. We conclude that 8-oxodG may be involved in mutations induced by NMOR plus UVA.

Effect of gamma irradiation on shelf life and bacterial and viral loads in hard-shelled clams (Mercenaria mercenaria).

The feasibility of using 60Co gamma irradiation to inactivate total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, and F-coliphage in hard-shelled clams, Mercenaria mercenaria, was investigated. The results of three trials indicated average D10 values of 1.32 kGy for total coliforms, 1.39 kGy for fecal coliforms, 1.54 kGy for E. coli, 2.71 kGy for C. perfringens, and 13.50 kGy for F-coliphage. Irradiation doses of > 0.5 kGy were significantly lethal to the shellfish.