F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

Seasonal detection of human viruses and coliphage in Newport Bay, California

Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in approximately 5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.     

Simple and rapid F+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination

Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters.     

Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Coliphages as indicators of enteroviruses.

Coliphages were monitored in conjunction with indicator bacteria and enteroviruses in a drinking-water plant modified to reduce trihalomethane production. Coliphages could be detected in the source water by direct inoculation, and sufficient coliphages were detected in enterovirus concentrates to permit following the coliphage levels through different water treatment processes. The recovery efficiency by different filter types ranged from 1 to 53%. Statistical analysis of the data indicated that enterovirus isolates were better correlated with coliphages than with total coliforms, fecal coliforms, fecal streptococci, or standard plate count organisms. Coliphages were not detected in finished water.     

Coliphages as indicators of enteroviruses

Coliphages were monitored in conjunction with indicator bacteria and enteroviruses in a drinking-water plant modified to reduce trihalomethane production. Coliphages could be detected in the source water by direct inoculation, and sufficient coliphages were detected in enterovirus concentrates to permit following the coliphage levels through different water treatment processes. The recovery efficiency by different filter types ranged from 1 to 53%. Statistical analysis of the data indicated that enterovirus isolates were better correlated with coliphages than with total coliforms, fecal coliforms, fecal streptococci, or standard plate count organisms. Coliphages were not detected in finished water.     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Seasonal Detection of Human Viruses and Coliphage in Newport Bay, California

Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in ~5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative Chlorine Resistance of Poliovirus I and Coliphages f2 and T2 in Water

Phage f2 is markedly more resistant to chlorine in water than either poliovirus or coliphage T(2). Its potential use as an indicator of viral pollution is suggested.     

Occurrence, source, and human infection potential of cryptosporidium and Giardia spp. in source and tap water in shanghai, china

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative.     

Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence

The contamination of beach waters occurs from the discharge of storm water and sanitary sewer over?ows containing faecal material. Additional faecal material derives from discharge of animals and waterfowl. In order to protect public from exposure to faecal‐contaminated water, it is required to test enteric indicators in beach water. The problem is that the traditional culture‐based methods cannot meet this goal because it takes too long (>24 h), so the results are not available until a day later. A rapid method for testing beach water for Escherichia coli within 1 h has been developed. Immunomagnetic separation (IMS) and ATP bioluminescence were used for selective capture and quanti?cation, respectively. This rapid method was compared to the current method (m‐TEC) using beach water samples. The beach samples were pre?ltered with a 20 µm pore size ?lter in order to remove algae, plant debris and large particles. The results showed that the pre?ltration step did not trap the bacteria which were present in the original water samples. The pre?ltered water was then passed through a 0.45 µm pore size ?lter for concentration. The deposited bacteria were resuspended and then mixed with superparamagnetic polystyrene beads (diameter of 0.6 µm) that were coated with E. coli antibodies. After IMS, the quanti?cation of the E. coli was done by ATP bioluminescence. The results obtained with IMS‐ATP bioluminescence correlated well with the plate count method (Rsq = 0.93). The detection limit of the assay was about 20 CFU/100 mL, which is well below the US EPA limits for recreational water. The entire procedure can be completed in less than 1 hour. The necessary equipment is portable and was tested on‐site. Copyright © 2004 John Wiley & Sons, Ltd.     

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.     

Evaluation of coliphage detection as a rapid indicator of water quality.

A rapid coliphage analysis technique for enumerating coliphages in natural waters has been evaluated by water quality laboratories located throughout the United States. Correlations were established between coliphages and coliforms in natural water systems. These correlations were highly significant. This relationship can thus be used to determine the number of fecal or total coliforms present in natural water samples based on an enumeration of coliphages. With this method, coliphages in natural water systems (containing greater than or equal to six coliphages per 100 ml) can be enumerated within 6 h.     

Bacterial Indicator Occurrence and the Use of an F+ Specific RNA Coliphage Assay to Identify Fecal Sources in Homosassa Springs, Florida

Abstract A microbiological water quality study of Homosassa Springs State Wildlife Park (HSSWP) and surrounding areas was undertaken. Samples were collected in November of 1997 (seven sites) and again in November of 1998 (nine sites). Fecal bacterial concentrations (total and fecal coliforms, Clostridium perfringens, and enterococci) were measured as relative indicators of fecal contamination. F⁺-specific coliphage genotyping was performed to determine the source of fecal contamination at the study sites. Bacterial levels were considerably higher at most sites in the 1997 sampling compared to the 1998 sampling, probably because of the greater rainfall that year. In November of 1997, 2 of the 7 sites were in violation of all indicator standards and guidance levels. In November of 1998, 1 of 9 sites was in violation of all indicator standard and guidance levels. The highest concentrations of all fecal indicators were found at a station downstream of the animal holding pens in HSSWP. The lowest levels of indicators were found at the Homosassa Main Spring vent. Levels of fecal indicators downstream of HSSWP (near the point of confluence with the river) were equivalent to those found in the Southeastern Fork and areas upstream of the park influences. F⁺ specific RNA coliphage analysis indicated that fecal contamination at all sites that tested positive was from animal sources (mammals and birds). These results suggest that animal (indigenous and those in HSSWP) and not human sources influenced microbial water quality in the area of Homosassa River covered by this study. Received: 12 May 1999; Accepted: 11 August 1999; Online Publication: 2 March 2000     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

The inactivation of bacteriophages infecting Bacteroides fragilis by chlorine treatment and UV-irradiation

Abstract The sensitivity to chlorination and to UV-irradiation of bacteriophage B40-8, which infects Bacteroides fragilis , was evaluated in comparison to that of Escherichia coli, Streptococcus faecalis , poliovirus 1, simian rotavirus 11 and coliphage f2. The results indicated that viruses persisted longer than bacteria in the presence of both disinfectants. Phage B40-8 was the most resistant microorganism to chlorination while coliphage f2 was the most resistant to UV-irradiation. In the latter, phage B40-8 was nevertheless as resistant as poliovirus and rotavirus. As expected, all microorganisms were more resistant to inactivation in sewage water than in tapwater.     

Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration.

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

PCR detection of pathogenic viruses in southern California urban rivers

AIMS: To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA. METHODS AND RESULTS: Three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR from 11 rivers and creeks. Faecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both faecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the quality of coastal water than following storm events. CONCLUSIONS: This study provides the first direct evidence that human viruses are prevalent in southern California urban rivers. Urban run-off impacts coastal water quality most significantly during the storm season. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health during water recreational activities, it is necessary to develop effective strategies to manage urban run-off during storm events.