F-RNA Coliphages Research Articles

Sunlight, season, snowmelt, storm, and source affect E. coli populations in an artificially ponded stream

Reducing fecal indicator bacteria, such as Escherichia coli ( E. coli), in streams is important for many downstream areas. E. coli concentrations within streams may be reduced by intervening ponds or wetlands through a number of physical and biological means. A section of Dunes Creek, a small coastal stream of southern Lake Michigan, was impounded and studied for 30 months from pre-through post-construction of the experimental pond. E. coli reduction became more predictable and effective with pond age. E. coli followed the hydrograph and increased several-fold during both rainfall and snowmelt events. Seasonally, the pond was more effective at reducing E. coli during summer than winter. Late summer, non-solar reduction or inactivation of E. coli in the pond was estimated at 72% and solar inactivation at 26%. E. coli DNA fingerprinting demonstrated that the winter population was genetically more homogeneous than the summer population. Detection of FRNA coliphages suggests that there was fecal contamination during heavy rain events. An understanding of how environmental factors interact with E. coli populations is important for assessing anticipated contaminant loading and the reduction of indicator bacteria in downstream reaches.

EVALUATION OF FUNG DOUBLE TUBE TEST FOR CLOSTRIDIUM PERFRINGENS AND EASYPHAGE TEST FOR F‐SPECIFIC RNA COLIPHAGES AS RAPID SCREENING TESTS FOR FECAL CONTAMINATION IN RECREATIONAL WATERS OF HAWAII

ABSTRACT The previously reported Fung double tube (FDT) for Clostridium perfringens and the newly developed EasyPhage test for F‐specific RNA coliphages were evaluated as screening tests for fecal contamination of fresh and marine recreational waters in Hawaii. These two tests were used to analyze water samples from marine swimming beaches, streams, nonswimming coastal waters and raw sewage. The major advantages of the FDT test include formation of anaerobic conditions without the need of anaerobic chamber systems and enumeration of colonies of C. perfringens in the tubes within 6 h. The results showed that 5 mL of water sample and 20 mL of 2× Shahidi Ferguson Perfringens medium were the best combination for the enumeration of C. perfringens in 5–6 h at 42C. This is a candidate, rapid beach monitoring method which can be used to close the beach on the same day the water samples were collected. The major advantages of the EasyPhage method include the rapid formation of gel without the use of heat, and addition of streptomycin and ampicillin to control interfering bacterial contamination. These two test methods allow most water laboratories to monitor environmental waters for C. perfringens and F‐specific RNA coliphages as indicators of fecal contamination.PRACTICAL APPLICATIONSMonitoring fecal contamination in water around the world is of great importance in public health. This paper presents rapid and efficient methods to monitor Clostridium perfringens and F‐specific RNA coliphages in waters in Hawaii, USA. The 6‐hour Fung‐Double Tube (FDT) system for enumeration of C. perfringens in water by use of SFP agar and incubation at 42C was perfected. The EasyPhage method by Scientific Methods, LLC (Grander, IN) for enumeration of FRNA coliphages in water using an ingenious nonthermal gelling system incubated for 12–16 h at 35C was tested. A Fung/Fujioka Scale (I = uncontaminated, acceptable, II = nonpoint contamination, III = sewage contamination, and VI = elevated sewage contamination, unacceptable) was developed to group the contamination level of recreational water for Hawaii. Forty‐six water samples were evaluated by both methods with excellent information concerning C. perfringens and FRNA phage in water in Hawaii.

Bacteriophage ecology in a small community sewer system related to their indicative role in sewage pollution of drinking water

In view of various studies looking for the merit of coliphages as indicators of water pollution with viruses originating from faecal material, a small agricultural community (population of approximately 1500 inhabitants of all ages, 2-3 km from Haifa) was selected in order to understand these bacteriophage ecology (F-RNA and somatic coliphages) in its sewer and oxidation pond system. Along the sewer lines, it was possible to isolate constantly both bacteriophage types (F-RNA and somatic coliphages) at 10(2)-10(4) plaque-forming units (pfu) ml(-1). The average numbers of somatic and F-RNA phages isolated from oxidation pond were 10(3)-10(4) pfu ml(-1); however, somatic coliphages were undetectable for several months (April-August). Significant high correlation (0.944 < R(2) < 0.99) was found between increased anionic detergent concentrations and F-RNA coliphage numbers. Infants less than 1 year old excreted both phage types and few only F-RNA coliphages (at high numbers > 10(5) pfu g(-1)) for up to 1 year. The excretion of F-RNA coliphages was highly linked to Escherichia coli F(+) harborage in the intestinal track as found in their faecal content. Finally, three bacterial hosts E. coli F(+), F(-) and CN(13) tested for survivability in sewage filtrate revealed that E. coli F(+) had the highest survivability under these conditions. Presence of somatic and F male-specific phages in sewer lines of a small community are influenced by several factors such as: anionic detergents, nutrients, temperature, source (mainly infants), shedding and survival capability of the host strain. Better understanding of coliphages ecology in sewer systems can enhance our evaluation of these proposed indicator/index microorganisms used in tracking environmental pollution of water, soil and crop contamination with faecal material containing enteric viruses.

Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques

Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because OPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination.

Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter

Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6×102 pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.

Peracetic acid (PAA) disinfection of primary, secondary and tertiary treated municipal wastewaters

The efficiency of peracetic acid (PAA) disinfection against enteric bacteria and viruses in municipal wastewaters was studied in pilot-scale. Disinfection pilot-plant was fed with the primary or secondary effluent of Kuopio municipal wastewater treatment plant or tertiary effluent from the pilot-scale dissolved air flotation (DAF) unit. Disinfectant doses ranged from 2 to 7 mg/l PAA in the secondary and tertiary effluents, and from 5 to 15 mg/l PAA in the primary effluents. Disinfection contact times were 4–27 min. Disinfection of secondary and tertiary effluents with 2–7 mg/l PAA and 27 min contact time achieved around 3 log reductions of total coliforms (TC) and enterococci (EC). PAA disinfection also significantly improved the hygienic quality of the primary effluents: 10–15 mg/l PAA achieved 3–4 log reductions of TC and EC, 5 mg/l PAA resulting in below 2 log reductions. F-RNA coliphages were more resistant against the PAA disinfection and around 1 log reductions of these enteric viruses were typically achieved in the disinfection treatments of the primary, secondary and tertiary effluents. Most of the microbial reductions occurred during the first 4–18 min of contact time, depending on the PAA dose and microorganism. The PAA disinfection efficiency remained relatively constant in the secondary and tertiary effluents, despite of small changes of wastewater quality (COD, SS, turbidity, 253.7 nm transmittance) or temperature. The disinfection efficiency clearly decreased in the primary effluents with substantially higher microbial, organic matter and suspended solids concentrations. The results demonstrated that PAA could be a good alternative disinfection method for elimination of enteric microbes from different wastewaters.

Use of reflectors to enhance the synergistic effects of solar heating and solar wavelengths to disinfect drinking water sources

Aluminum reflectors were added to solar units designed to inactivate faecal microorganisms (faecal coliform, E. coli, enterococci, FRNA coliphage, C. perfringens) in stream water and diluted sewage by the two mechanisms (solar heat, solar UV) known to inactivate microorganisms. During sunny conditions, solar units with and without reflectors inactivated E. coli to <1 CFU/100 ml to meet drinking water standards. Solar units with reflectors disinfected the water sooner by increasing the water temperature by 8-10 degrees C to 64-75 degrees C. However, FRNA coliphages were still detected in these samples, indicating that this treatment may not inactivate pathogenic human enteric viruses. During cloudy conditions, reflectors only increased the water temperature by 3-4 degrees C to a maximum of 43-49 degrees C and E. coli was not completely inactivated. Under sunny and cloudy conditions, the UV wavelengths of sunlight worked synergistically with increasing water temperatures and were able to disinfect microorganisms at temperatures (45-56 degrees C), which were not effective in inactivating microorganisms. Relative resistance to the solar disinfecting effects were C. perfringens > FRNA coliphages > enterococci > E. coli > faecal coliform.

Usefulness of monitoring tropical streams for male-specific RNA coliphages

The objective of this study was to evaluate the usefulness of monitoring streams in Hawaii for FRNA coliphages as a reliable indicator of sewage contamination. This study was undertaken as a result of our previous findings that monitoring streams in Hawaii for traditional faecal indicator bacteria (faecal coliform, Escherichia coli, enterococci) was not useful in determining when streams are contaminated with sewage, because environmental (soil) sources rather than sewage accounted for the high concentrations of faecal bacteria in streams. Two perennial streams, sewage and soil samples were monitored for traditional faecal indicator bacteria (faecal coliform, E. coli, enterococci) and FRNA coliphages. The results showed that sewage treatment processes and disinfection drastically reduced the concentrations of traditional faecal indicator bacteria but FRNA coliphages were still present in significant concentrations in the treated sewage effluents. These results indicate that monitoring sewage effluents and environmental waters for only traditional faecal indicator bacteria may not be adequately protective of human health effects. Ambient concentrations of traditional faecal indicator bacteria in soil and streams of Hawaii were consistently high but consistently low for FRNA coliphages, indicating that monitoring streams of Hawaii for FRNA coliphages can be used to determine when streams are contaminated with sewage.

Random survey of the microbial quality of bottled water in South Africa

Due to the increased demand and consumption of bottled water in South Africa, there has been a growing concern about the microbiological quality of this product. Retail outlets sell local as well as imported bottled water to consumers. The microbiological quality of 10 different (8 local and 2 imported) bottled water products were tested over a period of three months on days 1, 30 and 90. Tests for the detection of heterotrophic plate count (HPC) bacteria, total and faecal coliform bacteria, spore-forming Clostridium perfringens, somatic and F-RNA coliphages were performed on the samples. In addition samples were analysed for three selected enteric viruses, caliciviruses, enteroviruses and rotaviruses using the reverse transcriptase-polymerase chain reaction (RT-PCR). The results indicated that 8/10 of the bottled water samples analysed, met the requirements set by the South African Bureau of Standards (SABS) for HPCs in bottled water of less than 100 counts per m&#8467;. However, in two bottled water samples the average HPC bacteria counts were 2.64 x 102 cfu m&#8467;-1 and 8.89 x 103 cfu m&#8467;-1 respectively which exceeded the recommended SABS guideline. HPC counts showed a slight variation during the three-month period in the bottled water samples. Total and faecal coliform bacteria, enterococci, C. perfringens, bacteriophages or enteric viruses were not detected in any of the ten bottled water samples analysed. It can be concluded that the microbial quality of eight of the ten selected bottled water samples analysed was within the acceptable limits set by the SABS guidelines and therefore, was safe for human consumption. WaterSA Vol.30 (2) 2004: 203-210

Bacteriophages: update on application as models for viruses in water

Phages are valuable models or surrogates for enteric viruses because they share many fundamental properties and features. Amon g these are structure, composition, morphology, size and site of replication. Even though they use different host cells, colipha ges and Bacteroides fragilis phages predominantly replicate in the gastro-intestinal tract of humans and warm-blooded animals where enteric viruses also replicate. A major advantage of phages is that, compared to viruses, they are detectable by simple, inexp ensive and rapid techniques. In view of these features, phages are particularly useful as models to assess the behaviour and survival of enteric viruses in the environment, and as surrogates to assess the resistance of human viruses to water treatment and disinfec tion processes. Since there is no direct correlation between numbers of phages and viruses, phages cannot to a meaningful extent be used to indicate numbers of viruses in polluted water. The presence of phages typically associated with human and animal excre ta indicates the potential presence of enteric viruses. However, the absence of these phages from water environments is generally a meaningful indication of the absence of enteric viruses. This is because phages such as somatic coliphages, F-RNA coliphages a nd B. fragilis phages generally outnumber enteric viruses in water environments, and they are at least as resistant to unfavourable conditions including those in water treatment and disinfection processes. However, using highly sensitive molecular techniques viruses have been detected in drinking water supplies which yielded negative results in conventional tests for phages. Initial ly, data on phages were rather confusing because a wide variety of techniques was used. However, techniques for the detection of phages are being standardised internationally. This applies in particular to somatic and F-RNA coliphages, and B. fragilis phages, which are most commonly used in water quality assessment. Reliable and practical techniques now available include direct quantitativ e plaque assays on samples of water up to 100 ml, and qualitative tests on 500 m l or more using highly sensitive enrichment procedures.

Regrowth and survival of indicator microorganisms on the surfaces of household containers used for the storage of drinking water in rural communities of South Africa

The present study covered two rural communities of South Africa: Ncera and Ntselamanzi villages. Raw water from Ncera river is used by the community of Ncera village for drinking, while the community of Ntselamanzi receives their drinking water from Alice purification system. Treated water is supplied to the community by a public standpipe system. In rural communities of South Africa, many households use polyethylene (PE) and galvanized steel (GS) containers for the storage of their drinking water. To investigate the regrowth and survival of indicator microorganisms on the surface of household containers during the storage of drinking water, PE and GS slides were suspended in the appropriate household containers for a period of 48 h. This period of 48 h was chosen as the study period because results from the questionnaire indicated that the largest percentage (62%) of households store their water for that length of time. The experiment was performed to test drinking water as it is collected and stored by rural communities. No disinfection of household containers or slides was done during the study period. Attached coliphages (F-RNA (FP) and somatic phage (SP), coliform bacteria (total coliform (TC), presumptive Escherichia coli (EC), Salmonella (Sal) and Clostridium perfringens (CP) were measured during the study period. With the exception of CP , attached indicator microorganisms consisted of TC, presumptive E. coli and Salmonella, somatic and F-RNA coliphages, although the yield (average count) for the last four groups (EC: <1–3 cfu cm −2, Sal: <1–15 cfu cm −2, FP: <1–7 pfu cm −2, SP:<1–7 pfu cm −2) was lower than that of TC (3–183 cfu cm −2). However, the lowest yield of indicator microorganisms was noted for presumptive E. coli. Whereas the occurrence and survival of TC was noted on the surface of household containers during the entire period of the experimental study, other indicator microorganisms occurred from time to time. The regrowth of indicator microorganisms occurred 48 h after the exposure of slides to both types of test waters. This length of time mostly resulted in the regrowth of TC (with an increase in bacterial counts) while the persistence of other indicator organism groups on the surface of the slides was apparent. A comparison between PE and GS containers showed that more TC (average count) regrew on PE than on GS containers (for river water, PE: from 36 to 55 cfu cm −2, GS: from 25 to 26 cfu cm −2; for standpipe water, PE: from 147 to 183 cfu cm −2, GS from 3 to 4 cfu cm −2). This study revealed that both types of household containers supported the growth and survival of indicator microorganisms due to the bad quality of the intake water before storage. The storage of drinking water for 48 h mainly resulted in the regrowth of TC. Nevertheless, the persistence of other indicator microorganisms was observed on the surface of household containers.

New methods for the detection of viruses: call for review of drinking water quality guidelines

Drinking water supplies which meet international recommendations for source, treatment and disinfection were analysed. Viruses recovered from 100 L-1,000 L volumes by in-line glass wool filters were inoculated in parallel into four cell culture systems. Cell culture inoculation was used to isolate cytopathogenic viruses, amplify the nucleic acid of non-cytopathogenic viruses and confirm viability of viruses. Over a period of two years, viruses were detected in 23% of 413 drinking water samples and 73% of 224 raw water samples. Cytopathogenic viruses were detected in 6% raw water samples but not in any treated drinking water supplies. Enteroviruses were detected in 17% drinking water samples, adenoviruses in 4% and hepatitis A virus in 3%. In addition to these viruses, astro- and rotaviruses were detected in raw water. All drinking water supplies had heterotrophic plate counts of < 100/mL, total and faecal coliform counts of 0/100 mL and negative results in qualitative presence-absence tests for somatic and F-RNA coliphages (500 mL samples). These results call for a revision of water quality guidelines based on indicator organisms and vague reference to the absence of viruses.

Transport of bacteria and bacteriophages in irrigated effluent into and through an alluvial gravel aquifer

The movement of bacteria and bacteriophages into and through an alluvial gravel aquifer was investigated at a bordered strip effluent irrigation scheme near Christchurch, New Zealand. Irrigation of one set of strips resulted in the contamination, by faecal coliform bacteria, and somatic and F-RNA coliphages, of two bores, approximately 60 m and 445 m downstream of the centre of the strips. F-RNA coliphages showed the greatest attenuation between the soil surface and the first bore, and faecal coliforms the least. Estimates of percolation times through the 13 m vadoze zone (based on times to peak concentration in the groundwater) ranged from 1.6 to 10.5 hr, with travel times for the bacteriophages being 1.4–3.4 times longer than for the bacteria. Injection of oxidation pond effluent containing rhodamine WT dye into the first bore resulted in contamination of the second bore (385 m downstream) by the dye, F-RNA coliphages, and faecal coliforms. In a second experiment, injection (into the same bore) of a mixture of phage MS-2, the bacterial tracerEscherichia coli J6-2, and rhodamine WT dye, produced a similar result in the downstream bore and in a newly-installed bore, 401 m downstream. In both injection experiments, the phages exhibited the shortest times to peak concentrations in the downstream bore(s), followed by the bacteria, and then the dye. Attenuation of the bacteria and phages was similar, but the microbes exhibited 100-fold greater reduction than the dye. Flow direction and longitudinal dispersivity were determined in a preliminary analysis using an idealised 2-D dispersion model. This information, and other measured and reported data, were then used as inputs in a 3-D dispersion model. The predicted concentration curves were matched to the observed curves by trial and error adjustment of the decay constant (λ). The best curve fits were obtained with λ values higher than those reported elsewhere. It is suggested that many of the reported microbial decay values underestimate microbial reductions in groundwater because they do not account for other removal mechanisms, such as filtration, sedimentation and irreversible adsorption.

A simple membrane filtration-elution method for the enumeration of F-RNA, F-DNA and somatic coliphages in 100-ml water samples

A method which combines membrane filtration (MF) with a new swirling elution (SE) procedure was developed for the enumeration of F-RNA, F-DNA and somatic coliphages in 100 ml water samples. Using replicates of a diluted sewage seed, test methods were compared with overlay plate counts. The method (MF-SE) involves 3 min sample filtration through a membrane filter, 6 min swirling of the filter in 2 ml of eluent, followed by separate assay of the filter (face down on a host lawn) and eluent (by overlay plating). The MF and eluent plate counts are then combined. By altering eluent composition (beef extract + NaCl and/or Tween 80), the following maximum recoveries were obtained, using tap water and 0.45 μm pore size filters: 72.9% for F-RNA coliphages (on host Salmonella typhimurium WG49), 81.5% for F-DNA coliphages (on E. coli RR), and 100.5% for somatic coliphages (on Escherichia coli 13706/60). With 0.22 μm filters, the equivalent recovery rates were 75.5, 92.2 and 99.8%, respectively. Using the optimum combination of filter pore size and eluent composition, the equivalent recovery rates were 75.2, 77.1 and 85.1% from river water, and 45.9, 95.7 and 100.2% from sea water. It is concluded that the MF-SE method gives good recovery rates for all three phages from 100 ml samples of tap water and low turbidity fresh waters, good recoveries of F-DNA and somatic coliphages from sea water, but poor recovery of F-RNA coliphages from sea water.

Improved Specificity in Detecting F-Specific Coliphages in Environmental Samples by Suppression of Somatic Phages

Analysis of environmental waters for F-specific coliphages on the genetically constructed host Salmonella tvphimurium strain WG49 is hampered by the detection of Bomatic Salmonella phages. To improve the specificity of F-specific coliphage detection, two agents were tested for selective inhibition of Salmonella phage infectivity: heat-killed cells of female (F) S. tvphimurium WG45 and chemically extracted S. tvphimurium lipopolysaccharide (LPS). When both treatments were tested on pools of environmental isolates of Salmonella phages and the FRNA coliphage MS2, Salmonella phage infectivity was reduced by &amp;gt;90% with no change in MS2 detection. When environmental waters were tested by single agar layer or membrane filter methods, LPS increased the selectivity of F-specific coliphage detection from 28% to &amp;gt;90%, with no loss of sensitivity. The results of this study indicate that LPS effectively inhibits Bomatic Salmonella phage infectivity when added directly to a water sample or to molten agar medium or when used to treat filters on which phages have been concentrated.